Secondary literature sources for EFh

Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.

J Biol Chem. 2001; 276: 17762-9

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Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity ([Ca(2+)](0.5) of 25 and 83 &mgr;m in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.

Tescalcin, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, Col9a3, and renin are expressed in the mouse testis during the early stages of gonadal differentiation.

Endocrinology. 2001; 142: 455-63

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To identify genes that are differentially expressed in the developing testis we used representational difference analysis of complementary DNA from gonads of mouse embryos at 13.5 days postcoitum (dpc). Three genes were identified. One of them was a novel gene termed tescalcin that encoded a putative EF-hand Ca(2+)-binding protein. The open reading frame consisted of 642 nucleotides encoding a protein with 214 amino acids. Analysis of the predicted amino acid sequence revealed an N:-myristoylation motif and several phosphorylation sites in addition to an EF-hand Ca(2+)-binding domain. TESCALCIN: messenger RNA (mRNA) was present in fetal testis, but not in ovary or mesonephros, and was restricted to the testicular cords. Its expression was first detected in the male gonad at 11.5 dpc and demonstrated a pattern consistent with a role in the testis at the early stages of testis differentiation. Tescalcin is expressed in the testis of Kit(W/W-v) mice, indicating that it is not dependent on the presence of germ cells. The other two genes identified were collagen IX alpha3 (Col9a3) and RENIN: Col9a3 expression was present at low levels in male and female gonads at 11.5 dpc. Thereafter, it was markedly up-regulated in the male, but remained very low in the female. Expression of Col9a3 was restricted to testicular cords and was also detected in testis of Kit(W/W-v) mice. RENIN: mRNA was first detected in testis at 12.5 dpc, increased thereafter, and reached a peak at 16.5 dpc. RENIN: mRNA was localized in cells of the interstitium and cells at the border between the gonad and mesonephros. Expression of RENIN: in the ovary was not detected using standard conditions.

Molecular dynamics study of Ca(2+) binding loop variants of parvalbumin with modifications at the "gateway" position.

Protein Eng. 2001; 14: 115-26

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The helix-loop-helix (i.e. EF-hand) Ca(2+) ion binding motif is characteristic of a large family of high-affinity Ca(2+) ion binding proteins, including the parvalbumins and calmodulins. In this paper we describe a set of molecular dynamics computations on the major parvalbumin from the silver hake (SHPV-B). In all variants examined, both whole protein and fragments thereof, the ninth loop residue in the Ca(2+) binding coordination site in the CD helix-loop-helix region (the so-called "gateway" residue) has been mutated. The three gateway mutations examined are arginine, which has never been found at the gateway position of any EF-hand protein, cysteine, which is the residue observed least in natural EF-hand sites, and serine, which is the most common (by far) non-acidic residue substitution at this position in EF-hand proteins in general, but never in parvalbumins. Results of the molecular dynamics simulations indicate that all three modifications are disruptive to the integrity of the mutated Ca(2+) binding site in the whole parvalbumin protein. In contrast, only the arginine and cysteine mutations are disruptive to the integrity of the mutated Ca(2+) binding site in the CD fragment of the parvalbumin protein. Surprisingly, the serine variant of the CD helix-loop-helix fragment exhibited remarkable stability during the entire molecular dynamics simulation, with retention of the Ca(2+) binding site. These results indicate that there are no inherent problems (for Ca(2+) ion binding) associated with the sequence of the CD helix-loop-helix fragment that precludes the incorporation of serine at the gateway position. Since the CD site is totally disrupted in the whole protein serine variant, this indicates that the Ca(2+) ion binding deficiencies are most likely related to the unique interaction that exists between the paired EF-hands in the whole protein. Our theoretical results correlate well with previous studies on engineered EF-hand proteins and with all of our experimental evidence on the silver hake parvalbumin.

S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE).

FEBS Lett. 2001; 488: 85-90

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Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)-binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl(2) decreased the helical content by 27% whereas helicity was marginally increased by ZnCl(2). The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

J Am Chem Soc. 2001; 123: 2405-13

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The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

A novel EF-hand calcium-binding protein in the flagellum of the protozoan Tritrichomonas suis.

Parasitology. 2001; 122: 125-32

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The cloning and characterization of Ts-p41, an EF-hand calcium-binding protein of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA library was screened with monospecific antibodies affinity purified on an immunoreactive 41 kDa antigen in a Triton X-114 membrane-protein fraction. The resulting cDNA fragments turned out to be derived from 2 different genes encoding closely related Ts-p41 variants. The deduced amino acid sequences contained 6 EF-hand domains perfectly matching the canonical consensus motif and a putative C-terminal prenylation site. Northern and Southern hybridizations revealed that Ts-p41 was highly expressed and encoded by a gene-family. A cDNA encoding Ts-p41 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant Ts-p41 proteins bind Ca2+. In immunofluorescence, epitopes recognized by anti-Ts-p41 antibodies were distributed as well on the anterior flagella as on the recurrent flagellum of the parasite. Our findings with the parabasalid T. suis suggest that multiple EF-hand bearing calcium-binding proteins might be a common phenomenon associated with flagellar motility.

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy.

Am J Hum Genet. 2001; 69: 471-80

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Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.

Backbone dynamics of the calcium-signaling protein apo-S100B as determined by 15N NMR relaxation.

Biochemistry. 2001; 40: 3439-48

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Backbone dynamics of homodimeric apo-S100B were studied by (15)N nuclear magnetic resonance relaxation at 9.4 and 14.1 T. Longitudinal relaxation (T(1)), transverse relaxation (T(2)), and the (15)N-[(1)H] NOE were measured for 80 of 91 backbone amide groups. Internal motional parameters were determined from the relaxation data using the model-free formalism while accounting for diffusion anisotropy. Rotational diffusion of the symmetric homodimer has moderate but statistically significant prolate axial anisotropy (D( parallel)/D( perpendicular) = 1.15 +/- 0.02), a global correlation time of tau(m) = 7.80 +/- 0.03 ns, and a unique axis in the plane normal to the molecular symmetry axis. Of 29 residues at the dimer interface (helices 1 and 4), only one has measurable internal motion (Q71), and the order parameters of the remaining 28 were the highest in the protein (S(2) = 0.80 to 0.91). Order parameters in the typical EF hand calcium-binding loop (S(2) = 0.73 to 0.87) were slightly lower than in the pseudo-EF hand (S(2) = 0.75 to 0.89), and effective internal correlation times, tau(e), distinct from global tumbling, were detected in the calcium-binding loops. Helix 3, which undergoes a large, calcium-induced conformational change necessary for target-protein binding, does not show evidence of interchanging between the apo and Ca(2+)-bound orientations in the absence of calcium but has rapid motion in several residues throughout the helix (S(2) = 0.78 to 0.88; 10 < or = tau(e) < or = 30 ps). The lowest order parameters were found in the C-terminal tail (S(2) = 0.62 to 0.83). Large values for chemical exchange also occur in this loop and in regions nearby in space to the highly mobile C-terminal loop, consistent with exchange broadening effects observed.

Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I.

Biochemistry. 2001; 40: 10063-77

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The structure of the calcium-saturated C-domain of skeletal troponin C (CTnC) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (TnI) was determined using nuclear magnetic resonance (NMR) spectroscopy. The solution structure determined by NMR is similar to the structure of the C-domain from intact TnC in complex with TnI(1)(-)(47) determined by X-ray crystallography [Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. Changes in the dynamic properties of CTnC.2Ca2+ induced by Rp40 binding were investigated using backbone amide (15)N NMR relaxation measurements. Analysis of NMR relaxation data allows for extraction of motional order parameters on a per residue basis, from which the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy associated with complex formation can be estimated. The results indicate that binding of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix. The backbone conformational entropy change (-TDeltaS) associated with binding of Rp40 to CTnC.2Ca2+ determined from (15)N relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degrees C. However, estimation of thermodynamic quantities using a structural approach [Lavigne, P., Bagu, J. R., Boyko, R., Willard, L., Holmes, C. F., and Sykes, B. D. (2000) Protein Sci. 9, 252-264] reveals that the change in solvation entropy upon complex formation is dominant and overcomes the thermodynamic "cost" associated with "stiffening" of the protein backbone upon Rp40 binding. Additionally, backbone amide (15)N relaxation data measured at different concentrations of CTnC.2Ca2+.Rp40 reveal that the complex dimerizes in solution. Fitting of the apparent global rotational correlation time as a function of concentration to a monomer-dimer equilibrium yields a dimerization constant of approximately 8.3 mM.

[Effect of antibodies against S100 proteins on neural plasticity in sensitized and naive snails]

Zh Vyssh Nerv Deiat Im I P Pavlova. 2001; 51: 73-80

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The influence of antibodies against total S100 protein fraction (AB-S100) and S100b protein (AB-S100b) on the activity of LP11 and RP11 neurons were studied in naive snails and during the nociceptive sensitization. Application of AB-S100 or AB-S100b (0.1 mg/ml) initiated membrane depolarization, increase in its excitability, and depression of neural responses to sensory stimulation in nonsensitized snails. The sensitization produced facilitation of neural transmission and increase in membrane excitability. Exposure to AB-S100 or AB-S100b (0.1 mg/ml) during sensitization substantially reduced its effects on neural transmission and membrane excitability. The difference between the extent of synaptic facilitation in neurons of sensitized snails and neurons of snails sensitized under conditions of AB-S100 or AB-S100b application was comparable with synaptic depression in neurons of naive snails produced by the isolated application of AB-S100 or AB-S100b. Application of AB-S100 of AB-S100b in the dose of 0.01 mg/ml did not change the parameters of neural activity. The obtained evidence suggest that S100 proteins (in particular, S100b) in L-RP11 neurons are involved in the mechanisms of membrane excitability, regulation of membrane potential and synaptic transmission in naive snails and in the mechanisms of membrane plasticity in the neurons during development of nociceptive sensitization.

Calretinin (CR) is a neuronal EF-hand protein previously characterized as a calcium (micromolar affinity) binding protein. CR-containing neurons are spared in some neurodegenerative diseases, although it is as yet unconfirmed how CR plays an active role in this protection. Higher levels of some metal cations (e.g. copper and zinc) are associated with these diseases. At the same time, metals such as terbium (NMR and fluorescence) cadmium (NMR) and manganese (EPR) serve as useful calcium analogues in the study of EF-hand proteins. We survey the binding of the above-mentioned metal cations that might affect the structure and function of CR. Competitive 45Ca2+-overlay, competitive terbium fluorescence and intrinsic tryptophan fluorescence are used to detect the binding of metal cations to CR. Terbium and copper (half-maximal effect of 15 microM) bind to CR. Terbium has a similar or greater affinity for the calcium-binding sites of CR than calcium. Copper quenches the fluorescence of terbium-bound CR, and CR tryptophan residues and competes weakly for 45Ca2+-binding sites. Cadmium, magnesium, manganese and zinc bind less strongly (half-maximal effects above 0.1 mM). Therefore, only terbium appears to be a suitable analytical calcium analogue in further studies of CR. The principal conclusion of this work is that copper, in addition to calcium, might be a factor in the function of CR and a link between CR and neurodegenerative diseases.

Conformation and structural transitions in the EF-hands of calmodulin.

J Biomol Struct Dyn. 2001; 19: 47-57

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Calcium plays a key role in cellular signal transduction. Calmodulin, a protein binding four calcium ions, is found in all eukaryotic cells and is believed to activate such processes. The calcium binding loop found in this protein, the canonical EF-hand, is also found in a large number of other proteins such as troponins, parvalbumins, calbindins etc. Earlier analysis of the amino acid sequences of these proteins with a view of understanding evolution of protein families and signaling mechanisms have provided extensive evidence for a characteristic double gene duplication event in this family of proteins. These analyses have been extended here to the three dimensional structures and the biophysical properties of the sequence segments of calmodulin EF-hands. The clear evolutionary history that shows up in sequences is not reflected as clearly in the conformation of individual EF-hands, which may be a consequence of the much higher conservation pressure on the structure. Some evidence for the proposed gene duplication is implicit in the apo-holo structural transitions of the EF-hands. The profile of amino acid properties that might be significant for calcium binding, however, clearly reflects the gene duplication. These profiles might also provide insightful information on the calcium affinity of the EF-hand motifs and the nature of amino acid residues that constitute them.

Ca2+-independent binding of an EF-hand domain to a novel motif in the alpha-actinin-titin complex.

Nat Struct Biol. 2001; 8: 853-7

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The interaction between alpha-actinin and titin, two modular muscle proteins, is essential for sarcomere assembly. We have solved the solution structure of a complex between the calcium-insensitive C-terminal EF-hand domain of alpha-actinin-2 and the seventh Z-repeat of titin. The structure of the complex is in a semi-open conformation and closely resembles that of myosin light chains in their complexes with heavy chain IQ motifs. However, no IQ motif is present in the Z-repeat, suggesting that the semi-open conformation is a general structural solution for calcium-independent recognition of EF-hand domains.

The heterodimeric complex of MRP-8 (S100A8) and MRP-14 (S100A9). Antibody recognition, epitope definition and the implications for structure.

Eur J Biochem. 2001; 268: 353-63

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The S100 calcium-binding proteins MRP-8 (S100A8) and MRP-14 (S100A9) form a heterodimeric complex in the cytosol of monocyte and neutrophil cell types circulating in peripheral blood. This complex, but not the individual subunit proteins, is specifically recognized by mAb 27E10. Domains in MRP-8 and MRP-14 mediating heterodimeric complex formation have not yet been identified but it is predicted that the structure of the complex will be similar to homodimeric forms of other S100 proteins. This study makes use of the specificity of mAb 27E10, and an in vitro coupled transcription/translation system to further examine the formation and maintenance of the MRP-8/MRP-14 complex. Truncated mutants of MRP-14 that lack the N-terminal residues 1-4 or the extended C-terminal 'tail', both complex with MRP-8. These deleted domains of MRP-14 are therefore not essential for complex formation. Peptides from MRP-8 or MRP-14, used to induce the epitope recognized by mAb 27E10, show that a critical interaction in complex formation involves the N-terminal of MRP-8 interacting with MRP-14. Phage display analysis defined composite residues of the epitope recognized by mAb 27E10. The epitope is trans-subunit, composed of residues in the C-terminal ends of helix IV in MRP-14 and helix I of MRP-8. A further complex-specific mAb, named 5.5, recognizes the hydrophobic residues in helix IV of MRP-8, exposed during heterodimer formation. The definition of these two epitopes indicates that helices IV of MRP-8 and MRP-14 are also a prominent point of interaction and suggests that the subunit proteins will assume an antiparallel alignment in the heterodimer, similar in structure to the homodimeric forms of S100 proteins.

A calcium-binding protein with four EF-hand motifs in Streptomyces ambofaciens.

Biosci Biotechnol Biochem. 2001; 65: 156-60

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A gene (cabA) encoding a calcium-binding protein was cloned from Streptomyces ambofaciens. CabA was 180 amino acid residues long and contained four typical EF-hand motifs bearing high sequence similarity to the calcium-binding sites in calmodulin. Consistent with this, CabA showed distinct calcium-binding activity, comparable to bovine brain calmodulin. cabA was transcribed throughout growth, as found by S1 nuclease mapping. Southern hybridization experiments showed that a single copy of cabA was present in various Streptomyces species. A hypothetical relationship between CabA and aerial mycelium formation in this strain was examined, since S. ambofaciens showed calcium-dependent aerial mycelium formation. However, disruption of cabA or overexpression of cabA in S. ambofaciens caused no detectable phenotypic changes.

Solution structure calculations through self-orientation in a magnetic field of a cerium(III) substituted calcium-binding protein.

J Magn Reson. 2001; 148: 23-30

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Within the frame of a research aimed at characterizing paramagnetic metal ions capable of inducing self-orientation of metalloproteins in solution, we have studied the complex of the 75-amino-acid calcium-binding protein calbindin D(9k) with one Ce(III) ion (CaCeCb). Backbone (15)N-(1)H (1)J values have been determined for CaCeCb at two different magnetic fields. The above values showed a distinct dependence on the magnetic field, which is caused by the partial orientation of the molecule in solution. The difference in the values at the two magnetic fields provides structural constraints, which have been used to refine the structure of CaCeCb. The refined structure showed an improvement in terms of the number of residues falling in favored regions of the Ramachandran plot. The comparison of the molecular magnetic susceptibility tensor, obtained from the (15)N-(1)H (1)J values, with the magnetic susceptibility tensor of the metal, obtained from pseudocontact shifts, showed that the orientation of the molecule in solution is mainly determined by the Ce(III) ion. This paper shows that Ce(III), like low-spin Fe(III) in hemoproteins, is sufficiently magnetically anisotropic to induce self-orientation to an extent which can be exploited for solution structure determination. Copyright 2001 Academic Press.

We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.

Characterization of apo and partially saturated states of calerythrin, an EF-hand protein from S. erythraea: a molten globule when deprived of Ca(2+).

Protein Sci. 2001; 10: 74-82

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Calerythrin, a four-EF-hand calcium-binding protein from Saccharopolyspora erythraea, exists in an equilibrium between ordered and less ordered states with slow exchange kinetics when deprived of Ca(2+) and at low temperatures, as observed by NMR. As the temperature is raised, signal dispersion in NMR spectra reduces, and intensity of near-UV CD bands decreases. Yet far-UV CD spectra indicate only a small decrease in the amount of secondary structure, and SAXS data show that no significant change occurs in the overall size and shape of the protein. Thus, at elevated temperatures, the equilibrium is shifted toward a state with characteristics of a molten globule. The fully structured state is reached by Ca(2+)-titration. Calcium first binds cooperatively to the C-terminal sites 3 and 4 and then to the N-terminal site 1, which is paired with an atypical, nonbinding site 2. EF-hand 2 still folds together with the C-terminal half of the protein, as deduced from the order of appearance of backbone amide cross peaks in the NMR spectra of partially Ca(2+)-saturated states.

Among the EF-hand Ca(2+)-binding proteins, parvalbumin (PV) and calbindin D9k (CaB) have the function of Ca(2+) buffers. They evolved from an ancestor protein through two phylogenetic pathways, keeping one pair of EF-hands. They differ by the extra helix-loop-helix (AB domain) found in PV and by the linker between the binding sites. To investigate whether the deletion of AB in PV restores a CaB-like structure, we prepared and solved the structure of the truncated rat PV (PVratDelta37) by X-ray and NMR. PVratDelta37 keeps the PV fold, but is more compact, having a well-structured linker, which differs remarkably from CaB. PvratDelta37 has no stable apo-form, has lower affinity for Ca(2+) than full-length PV, and does not bind Mg(2+), in contrast to CaB. Structural differences of the hydrophobic core are partially responsible for lowering the calcium-binding affinity of the truncated protein. It can be concluded that the AB domain, like the linker of CaB, plays a role in structural stabilization. The AB domain of PV protects the hydrophobic core, and is required to maintain high affinity for divalent cation binding. Therefore, the AB domain possibly modulates PV buffer function. PVratDelta37 (Type PDB; Value 1G33; Service) Proteins 2001;45:117-128. Copyright 2001 Wiley-Liss, Inc.

The structure of calbindin D(9k) with two substitutions was determined by X-ray crystallography at 1.8-A resolution. Unlike wild-type calbindin D(9k), which is a monomeric protein with two EF-hands, the structure of the mutated calbindin D(9k) reveals an intertwined dimer. In the dimer, two EF-hands of the monomers have exchanged places, and thus a 3D domain-swapped dimer has been formed. EF-hand I of molecule A is packed toward EF-hand II of molecule B and vice versa. The formation of a hydrophobic cluster, in a region linking the EF-hands, promotes the conversion of monomers to 3D domain-swapped dimers. We propose a mechanism by which domain swapping takes place via the apo form of calbindin D(9k). Once formed, the calbindin D(9k) dimers are remarkably stable, as with even larger misfolded aggregates like amyloids. Thus calbindin D(9k) dimers cannot be converted to monomers by dilution. However, heating can be used for conversion, indicating high energy barriers separating monomers from dimers.

Calmodulin and other members of the EF-hand protein family are known to undergo major changes in conformation upon binding Ca(2+). However, some EF-hand proteins, such as calbindin D9k, bind Ca(2+) without a significant change in conformation. Here, we show the importance of a precise balance of solvation energetics to conformational change, using mutational analysis of partially buried polar groups in the N-terminal domain of calmodulin (N-cam). Several variants were characterized using fluorescence, circular dichroism, and NMR spectroscopy. Strikingly, the replacement of polar side chains glutamine and lysine at positions 41 and 75 with nonpolar side chains leads to dramatic enhancement of the stability of the Ca(2+)-free state, a corresponding decrease in Ca(2+)-binding affinity, and an apparent loss of ability to change conformation to the open form. The results suggest a paradigm for conformational change in which energetic strain is accumulated in one state in order to modulate the energetics of change to the alternative state.

By photoaffinity labeling with a tritiated azido derivative of phenylarsine oxide (PAO), 4[N-(4-azido-2-nitrophenyl)amino-[(3)H]acetamido]phenylarsine oxide ([(3)H]azidoPAO), we demonstrate that PAO binds selectively to the S100 A8/A9 complex of bovine neutrophil cytosol (previously known as p7/p23, homologous to the MRP-8/MRP-14 complex of human phagocytes). Using a semirecombinant cell free assay of oxidase activation and the determination of oxidase activity by the production of the superoxide anion O(-)(2), we found that the PAO binding protein (p7/p23) was able to potentiate the activation of NADH oxidase and that this effect was synergized by PAO. The p7/p23 protein complex of bovine neutrophils can therefore be considered as a positive regulator of NADPH oxidase activation in neutrophils. Copyright 2001 Academic Press.

Structure of Escherichia coli fragment TR2C from calmodulin to 1.7 A resolution.

Acta Crystallogr D Biol Crystallogr. 2001; 57: 664-9

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Fragment TR2C is the C-terminal part of the calcium-binding protein calmodulin, including residues 78-148. The crystal structure of TR2C was solved by molecular replacement and refined to a conventional R value of 21.8% (R(free) = 22.0%), using all data in the resolution range 20.0-1.7 A. This study shows that the secondary structure of TR2C, a pair of EF-hand motifs with two calcium-binding sites, is similar to the corresponding motifs in intact calmodulin. However, it also indicates that the N-terminus of helix E is closer to the C-terminus of helix H in TR2C than in the intact protein and that the loop connecting the EF-hands shows different conformations in the two structures. The crystal structure of TR2C was further found to be similar to the set of NMR structures of this fragment, although some pronounced differences exist.

Role of the structural domain of troponin C in muscle regulation: NMR studies of Ca2+ binding and subsequent interactions with regions 1-40 and 96-115 of troponin I.

Biochemistry. 2000; 39: 2902-11

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The interaction between the calcium binding and inhibitory components of troponin is central to the regulation of muscle contraction. In this work, two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-?1H,15N?-HSQC NMR) spectroscopy was used to determine the stoichiometry, affinity, and mechanisms for binding of Ca2+ and two synthetic TnI peptides [TnI1-40 (or Rp40) and TnI96-115] to the isolated C-domain of skeletal troponin C (CTnC). The Ca2+ titration revealed that 2 equiv of Ca2+ binds to sites III and IV of CTnC with strong positive cooperativity and high affinity [dissociation constant (KD) = 0.1 microM]. In this process, CTnC folds from a largely unstructured state to a compact domain capable of interacting with TnI. Titration of CTnC x 2Ca2+ with Rp40 occurs with a 1:1 stoichiometry and a KD of 2 +/- 1 microM. Titration of CTnC x 2Ca2+ with a peptide corresponding to the inhibitory region of TnI (TnI96-115) also reveals a 1:1 ratio, but weaker affinity (KD = 47 +/- 7 microM). Both Rp40- and TnI96-115-induced backbone amide chemical shift changes of CTnC x 2Ca2+ are similarly distributed along the sequence, indicating that these two regions of TnI may compete for the same binding site on CTnC x 2Ca2+. The changes induced by Rp40 are much larger, however, and define the interaction sites on TnC and regions where the flexibility of hinge and terminal residues is altered. To investigate the possibility of direct competition, TnI(96-115) was titrated into the CTnC x 2Ca(2+) x Rp40 complex, whereas Rp40 was titrated into the CTnC x 2Ca2+. TnI96-115 complex. The results show that Rp40 can displace TnI96-115 completely, while TnI96-115 has no effect on CTnC x 2Ca2+ x Rp40. Recent proposals for the mechanism of muscle regulation [Tripet, B. P., Van Eyk, J. E., and Hodges, R. S. (1997) J. Mol. Biol. 271, 728-750] suggest that the N-terminal and inhibitory regions of TnI competitively bind the structural domain of TnC. The findings presented here indicate that additional factors, such as interactions between the N-domain of TnC with the C-domain of TnI or the C-domain of TnT, are required, if the inhibitory region is going to successfully compete for the structural domain of TnC.

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

Effects of mutations in the calcium-binding sites of recoverin on its calcium affinity: evidence for successive filling of the calcium binding sites.

Protein Eng. 2000; 13: 783-90

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A molecule of the photoreceptor Ca(2+)-binding protein recoverin contains four potential EF-hand Ca(2+)-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca(2+)-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein's thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca(2+)-binding constants according to the sequential binding scheme gave the values 3.7 x 10(6) and 3.1 x 10(5) M(-1) for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form.

Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein.

J Biol Chem. 2000; 275: 31178-82

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Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells. A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J. (1998) J. Neurochem. 70, 1793-1798). Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP. A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin. CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% alpha-helix, 15% beta -sheet, and 81% random coil. Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 microm for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions. NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of (15)N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

The CREC family, a novel family of multiple EF-hand, low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells.

FEBS Lett. 2000; 466: 11-8

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The CREC family consists of a number of recently discovered multiple (up to seven) EF-hand proteins that localise to the secretory pathway of mammalian cells. At present, the family includes reticulocalbin, ERC-55/TCBP-49/E6BP, Cab45, calumenin and crocalbin/CBP-50. Similar proteins are found in quite diverse invertebrate organisms such as DCB-45 and SCF in Drosophila melanogaster, SCF in Bombyx mori, CCB-39 in Caenorhabditis elegans and Pfs40/PfERC in Plasmodium falciparum. The Ca(2+) affinity is rather low with dissociation constants around 10(-4)-10(-3) M. The proteins may participate in Ca(2+)-regulated activities. Recent evidence has been obtained that some CREC family members are involved in pathological activities such as malignant cell transformation, mediation of the toxic effects of snake venom toxins and putative participation in amyloid formation.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.

Crystal structure of human grancalcin, a member of the penta-EF-hand protein family.

J Mol Biol. 2000; 300: 1271-81

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Grancalcin is a Ca(2+)-binding protein expressed at high level in neutrophils. It belongs to the PEF family, proteins containing five EF-hand motifs and which are known to associate with membranes in Ca(2+)-dependent manner. Prototypic members of this family are Ca(2+)-binding domains of calpain. Our recent finding that grancalcin interacts with L-plastin, a protein known to have actin bundling activity, suggests that grancalcin may play a role in regulation of adherence and migration of neutrophils. The structure of human grancalcin has been determined at 1.9 A resolution in the absence of calcium (R-factor of 0.212 and R-free of 0.249) and at 2. 5 A resolution in the presence of calcium (R-factor of 0.226 and R-free of 0.281). The molecule is predominantly alpha-helical: it contains eight alpha-helices and only two short stretches of two-stranded beta-sheets between the loops of paired EF-hands. Grancalcin forms dimers through the association of the unpaired EF5 hands in a manner similar to that observed in calpain, confirming this mode of association as a paradigm for the PEF family. Only one Ca(2+) was found per dimer under crystallization conditions that included CaCl(2). This cation binds to EF3 in one molecule, while this site in the second molecule of the dimer is unoccupied. This unoccupied site shows higher mobility. The structure determined in the presence of calcium, although does not represent a fully Ca(2+)-loaded form, suggests that calcium induces rather small conformational rearrangements. Comparison with calpain suggests further that the relatively small magnitude of conformational changes invoked by calcium alone may be a characteristic feature of the PEF family. Moreover, the largest differences are localized to the EF1, thus supporting the notion that calcium signaling occurs through this portion of the molecule and that it may involve the N-terminal Gly/Pro rich segment. Electrostatic potential distribution shows significant differences between grancalcin and calpain domain VI demonstrating their distinct character.

Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1-132 and 133-261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133-261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1-132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1-132 and 133-261 fragments. The affinity between these two fragments is 2 x 10(7) M(-1), with association and dissociation rate constants of 2.7 x 10(4) M(-1) s(-1) and 1.4 x 10(-3) s(-1), respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual alpha-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The alpha-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.

Heterocomplex formation between metastasis-related protein S100A4 (Mts1) and S100A1 as revealed by the yeast two-hybrid system.

FEBS Lett. 2000; 475: 187-91

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S100A4 (Mts1) is a Ca(2+)-binding protein of the S100 family. This protein plays an important role in promoting tumor metastasis. In order to identify S100A4 interacting proteins, we have applied the yeast two-hybrid system as an in vivo approach. By screening a mouse mammary adenocarcinoma library, we have demonstrated that S100A4 forms a heterocomplex with S100A1, another member of the S100 family. The non-covalent heterodimerization was confirmed by fluorescence spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Mutational analysis revealed that replacement of Cys(76) and/or Cys(81) of S100A4 by Ser abolishes the S100A4/S100A1 heterodimerization, but does not affect the S100A4 homodimerization in vivo.

The Ca2 calbindin protein in which one calcium has been substituted with Ce(III), Yb(III) and Dy(III) displays substantial alignment in high magnetic fields due to the high anisotropy of the metal magnetic susceptibility. This property has allowed the measurement of residual dipolar coupling contributions to 1J(HN) and 2J(HH) couplings of asparagine and glutamine NH2 moieties. Such data have been used to aid structural characterization of these groups. The exploitation of auto-orientation of magnetic anisotropic metalloproteins represents a step ahead in the investigation of the conformational space of peripheral residues that are not fixed by the protein folding.

Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca(2+). We have determined the crystal structure of cTnC, with three bound Ca(2+) ions, complexed with the calcium-sensitizer bepridil, to 2.15-A resolution. In contrast to apo- and 3Ca(2+)-cTnC, the drug-bound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca(2+)-sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147-163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca(2+)-sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca(2+) regulation by troponin C in cardiac muscle.

The cooperative binding of Ca2+ ions is an essential functional property of the EF-hand family of Ca2+-binding proteins. To understand how these proteins function, it is essential to characterize intermediate binding states in addition to the apo- and holo-proteins. The three-dimensional solution structure and fast time scale internal motional dynamics of the backbone have been determined for the half-saturated state of the N56A mutant of calbindin D9k with Ca2+ bound only in the N-terminal site. The extent of conformational reorganization and a loss of flexibility in the C-terminal EF-hand upon binding of an ion in the N-terminal EF-hand provide clear evidence of the importance of site-site interactions in this family of proteins, and demonstrates the strength of long-range effects in the cooperative EF-hand Ca2+-binding domain.

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Calbindin is a small (75 residues) helix-loop-helix ("EF-hand") calcium-binding protein belonging to the calmodulin superfamily. It binds two Ca(2+) ions. Continuum electrostatics in combination with the boundary element method was employed for the calculation of the acid-dissociation constants K(a) (pK(a) = -log K(a)) values of all titratable residues in the protein. The objectives were to determine quantitatively the effects of divalent ion binding and small ion-induced structural changes on predicted pK(a)'s. Computations were carried out for the apo and holo form of calbindin, for which both X-ray and NMR structures were available. Comparison was made with several sets of experimental pK(a) values determined by NMR spectroscopy. Different choices of the dielectric constant (ranging from 4 to 78.5) for calbindin and variations in ionic strength (from 0 to 0.3 M) were investigated in a systematic fashion. Removal of the two bound Ca(2+) ions increases the pK(a) values of all residues if no conformational changes were allowed. If conformational differences between the apo and holo were accounted for, shifts in either direction were observed. Titrating groups that are directly involved in Ca(2+) binding (Asp and Glu) required a dielectric constant of 78.5 for the holo structure to obtain a reasonable estimate of their pK(a)'s. For the apo structure, passable values for the pK(a)'s of these ligating groups could be determined if the structure was allowed to relax upon ion removal.

The identification of a putative apical Ca++ channel in 1,25dihydroxyvitamin D3 responsive epithelia (proximal intestine and the distal nephron) as well as recent studies using calbindin-D28k knock-out mice indicating the first direct in-vivo evidence for a role for this calcium-binding protein in renal calcium absorption suggest mechanisms, which had remained incomplete, related to the control of renal calcium absorption.

Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations.

J Biol Chem. 2000; 275: 35106-15

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To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90). Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control. Circular dichroism measurements of wild-type N domain as a function of pCa (= -log[Ca(2+)]) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ([theta](222 nm)) could be assigned to site II Ca(2+) binding. With E41A, E77A, and cardiac TnC N domains this [theta](222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+). Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs. Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.

[Use of method of protein engineering in studying calcium-binding proteins]

Biofizika. 2000; 45: 990-1006

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Major results of the use of protein engineering methods in studies of calcium-binding proteins with the highest affinity for calcium and known three-dimensional structure (parvalbumin, calmodulin, troponin C, calbindin, recoverin, alpha-lactalbumin, and others) are presented. Specific features of recombinant calcium-binding proteins are discussed. Experiments with genetic introduction of fluorescent probes, tryptophan and tyrosine, into proteins are overviewed. Effects of mutations in different parts of protein molecules (calcium-binding loops, hydrophobic core, and others) on their structure and properties and attempts of creation of artificial calcium-binding sites are discussed.

Calcium-induced flexibility changes in the troponin C-troponin I complex.

Biochim Biophys Acta. 2000; 1479: 247-54

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The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.

Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica.

FEBS Lett. 2000; 486: 112-6

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We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.

Cardiac troponin I(129-149) binds to the calcium saturated cardiac troponin C/troponin I(1-80) complex at two distinct sites. Binding of the first equivalent of troponin I(129-149) was found to primarily affect amide proton chemical shifts in the regulatory domain, while the second equivalent perturbed amide proton chemical shifts within the D/E linker region. Nitrogen-15 transverse relaxation rates showed that binding the first equivalent of inhibitory peptide to the regulatory domain decreased conformational exchange in defunct calcium binding site I and that addition of the second equivalent of inhibitory peptide decreased flexibility in the D/E linker region. No interactions between the inhibitory peptide and the C-domain of cardiac troponin C were detected by these methods demonstrating that the inhibitory peptide cannot displace cTnI(1-80) from the C-domain.

Understanding the process of Ca(2+)/Mg(2+)exchange during muscle excitation and relaxation is fundamental to elucidating the mechanism of Ca(2+)-regulated muscle contraction. During the resting phase, the C-domain of cardiac troponin C may be occupied by either Ca(2+)or Mg(2+). Here, complexes of recombinant cardiac troponin C(81-161) and the N terminus of cardiac troponin I, representing residues 33-80, were generated in the presence of saturating Mg(2+). Heteronuclear multi-dimensional nuclear magnetic resonance experiments were used to obtain backbone assignments of the Mg(2+)-loaded complex. In the presence of cardiac troponin I, the affinity of site IV for Mg(2+)is increased. Comparison of Mg(2+)and Ca(2+)-loaded complexes reveals that chemical shift differences are primarily localized to metal-binding sites III and IV, defining positions within these sites that have distinct Ca(2+)/Mg(2+)conformations. The observed transition from the Mg(2+)-loaded to Ca(2+)-loaded form demonstrates that sites III and IV fill simultaneously with Ca(2+)displacing Mg(2+). However, even in the absence of excess Ca(2+), Mg(2+)does not readily displace Ca(2+)in the isolated binary complex. Thus, the Mg(2+)-loaded conformer may only represent a small fraction of the total cardiac troponin C found in the sarcomere.

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.

Studies of the role of the integrin EF-hand, Ca2+-binding sites in glycosylphosphatidylinositol-specific phospholipase D: reduced expression following mutagenesis of residues predicted to bind Ca2+.

Arch Biochem Biophys. 1999; 361: 142-8

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Previous studies of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) have demonstrated that GPI-PLD can bind Ca2+ ions with high specificity (J.-Y. Li, K. Hollfelder, K.-S. Huang, and M. G. Low, J. Biol. Chem. 269, 28063-28971, 1994). In this study the functional role of the bound Ca2+ ions was evaluated. The enzymatic activity of purified GPI-PLD, which was depleted of divalent cations by pretreatment with EDTA, EGTA, or 1, 10-phenanthroline, could be completely restored with Zn2+ (and partially with Co2+), which indicates that Ca2+ can be removed from the protein without affecting its enzymatic activity. This result suggested that Ca2+ bound to GPI-PLD has a structural or regulatory role but is not required for GPI hydrolysis. To evaluate these possibilities we transfected COS cells with GPI-PLD mutants in which the predicted Ca2+-binding sites were either deleted completely or altered by single-residue substitution. All of the mutations showed substantial reductions in the amount of GPI-PLD secreted into the medium (0-6% of wild type). The data indicate that bound Ca2+ plays an important role in the initial folding, intracellular transport, or secretion of GPI-PLD even though it has no discernible role in the mature, secreted protein.

Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains.

Protein Expr Purif. 1999; 17: 465-76

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Calretinin (CR) is a calcium-binding, neuronal protein of undefined function. Related proteins either buffer intracellular calcium concentrations or are involved in calcium-signaling pathways. We transformed three CR gene fragment sequences, corresponding to its three complementary domains (I-II, III-IV, and V-VI), into Pichia pastoris. High yields of extracellular expression, of more than 200 mg/liter, were achieved. Simple purification protocols provide high yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose chromatography for domain V-VI. To our knowledge, this is the first report of the expression of an EF-hand protein using P. pastoris. Direct comparison of the purified yields of domain I-II indicates a approximately 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies recognized the appropriate domains. All three CR domains bind (45)Ca and the domain containing EF-hands V and VI seems to have a lower calcium capacity than the other domains. Circular dichroism indicates a high helix content for each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous studies, while tyrosine emission fluorescence indicates calcium-induced structural changes also occur in domain V-VI. The methods and expression levels achieved are suitable for future NMR labeling of the proteins, with (15)N and (13)C, and structure-function studies that will help to further understand CR function.

Two efficient procedures for large-scale purification of calbindin D9k from porcine intestine by HPLC were developed. Both protocols start with heat treatment of the intestinal tissue followed by acetic acid extraction, a capture with alginic acid, NaCl precipitation of other proteins, and a concentration step on Amberlite XAD-2. In the first method, a single reverse-phase HPLC step completes the purification and results in milligram quantities of pure calbindin. In the second method, an additional ion exchange HPLC step was introduced, followed by a reverse-phase HPLC resulting in 100 milligram-scale preparations of homogeneous calbindin in a 56% yield from the Amberlite step. Both methods yielded a homogeneous metal-free apoprotein with a molecular weight of 8838.0 +/- 8.8 as analyzed by MALDI TOF mass spectrometry corresponding to N-acetylated porcine calbindin. The isolated apocalbindin was fully reconstituted with 2 molar equivalents of Ca(2+) and the protein displayed UV and fluorescence spectra characteristic of those of native calbindin D9k.

The use of dipolar couplings for determining the solution structure of rat apo-S100B(betabeta).

Protein Sci. 1999; 8: 800-9

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The relative orientations of adjacent structural elements without many well-defined NOE contacts between them are typically poorly defined in NMR structures. For apo-S100B(betabeta) and the structurally homologous protein calcyclin, the solution structures determined by conventional NMR exhibited considerable differences and made it impossible to draw unambiguous conclusions regarding the Ca2+-induced conformational change required for target protein binding. The structure of rat apo-S100B(betabeta) was recalculated using a large number of constraints derived from dipolar couplings that were measured in a dilute liquid crystalline phase. The dipolar couplings orient bond vectors relative to a single-axis system, and thereby remove much of the uncertainty in NOE-based structures. The structure of apo-S100B(betabeta) indicates a minimal change in the first, pseudo-EF-hand Ca2+ binding site, but a large reorientation of helix 3 in the second, classical EF-hand upon Ca2+ binding.

The crystal structure of calcium-bound unmyristoylated bovine neurocalcin from Escherichia coli has been determined at 2.4 A resolution. The three-dimensional structure reveals a highly compact structure consisting of: (i) two pairs of calcium-binding EF-hands (EF1-EF2 and EF3-EF4); (ii) a calcium ion bound at EF2, EF3 and EF4 sites; and (iii) an EF1-hand that is disabled from calcium-binding due to a Cys-Pro sequence in the Ca2+-binding loop. The crystal structure of neurocalcin resembles photoreceptor recoverin in overall topology, however its EF2- and EF4-hands differ. Recently, neurocalcin in the calcium-bound state has been shown to stimulate mammalian rod outer segment membrane guanylate cyclase. A possible site for cyclase activity based on the three-dimensional structure is discussed.

Characterization of a B cell surface antigen with homology to the S100 protein MRP8.

Biochem Biophys Res Commun. 1999; 263: 17-22

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The S100 proteins comprise a large sub-family of the EF-hand calcium-binding proteins. Here we describe a novel monoclonal antibody recognizing a B cell surface antigen. This monoclonal antibody immunoprecipitates three proteins in the 12-18 kDa range and the smallest of these proteins has a striking homology at its amino-terminus to human MRP8, a myeloid specific member of the S100 family. Similarly to MRP8 in myeloid cells, this antigen is expressed in the cytoplasm of B cells and is secreted by LPS-induced activated B cells. This surface antigen is not B cell specific. Since MRP8 is not expressed by lymphoid cells, however, this antibody appears to recognize a new member of the S100 family.

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

Nat Struct Biol. 1999; 6: 89-95

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The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

BACKGROUND: The EF-hand family is a large set of Ca(2+)-binding proteins that contain characteristic helix-loop-helix binding motifs that are highly conserved in sequence. Members of this family include parvalbumin and many prominent regulatory proteins such as calmodulin and troponin C. EF-hand proteins are involved in a variety of physiological processes including cell-cycle regulation, second messenger production, muscle contraction, microtubule organization and vision. RESULTS: We have determined the structures of parvalbumin mutants designed to explore the role of the last coordinating residue of the Ca(2+)-binding loop. An E101D substitution has been made in the parvalbumin EF site. The substitution decreases the Ca(2+)-binding affinity 100-fold and increases the Mg(2+)-binding affinity 10-fold. Both the Ca(2+)- and Mg(2+)-bound structures have been determined, and a structural basis has been proposed for the metal-ion-binding properties. CONCLUSIONS: The E101D mutation does not affect the Mg(2+) coordination geometry of the binding loop, but it does pull the F helix 1.1 A towards the loop. The E101D-Ca(2+) structure reveals that this mutant cannot obtain the sevenfold coordination preferred by Ca(2+), presumably because of strain limits imposed by tertiary structure. Analysis of these results relative to previously reported structural information supports a model wherein the characteristics of the last coordinating residue and the plasticity of the Ca(2+)-binding loop delimit the allowable geometries for the coordinating sphere.

Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.

Biochemistry. 1999; 38: 2-10

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In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.

A multigenic family of Ca2+-binding proteins of the EF-hand type known as S100 comprises 19 members that are differentially expressed in a large number of cell types. Members of this protein family have been implicated in the Ca2+-dependent (and, in some cases, Zn2+- or Cu2+-dependent) regulation of a variety of intracellular activities such as protein phosphorylation, enzyme activities, cell proliferation (including neoplastic transformation) and differentiation, the dynamics of cytoskeleton constituents, the structural organization of membranes, intracellular Ca2+ homeostasis, inflammation, and in protection from oxidative cell damage. Some S100 members are released or secreted into the extracellular space and exert trophic or toxic effects depending on their concentration, act as chemoattractants for leukocytes, modulate cell proliferation, or regulate macrophage activation. Structural data suggest that many S100 members exist within cells as dimers in which the two monomers are related by a two-fold axis of rotation and that Ca2+ binding induces in individual monomers the exposure of a binding surface with which S100 dimers are believed to interact with their target proteins. Thus, any S100 dimer is suggested to expose two binding surfaces on opposite sides, which renders homodimeric S100 proteins ideal for crossbridging two homologous or heterologous target proteins. Although in some cases different S100 proteins share their target proteins, in most cases a high degree of target specificity has been described, suggesting that individual S100 members might be implicated in the regulation of specific activities. On the other hand, the relatively large number of target proteins identified for a single S100 protein might depend on the specific role played by the individual regions that in an S100 molecule contribute to the formation of the binding surface. The pleiotropic roles played by S100 members, the identification of S100 target proteins, the analysis of functional correlates of S100-target protein interactions, and the elucidation of the three-dimensional structure of some S100 members have greatly increased the interest in S100 proteins and our knowledge of S100 protein biology in the last few years. S100 proteins probably are an example of calcium-modulated, regulatory proteins that intervene in the fine tuning of a relatively large number of specific intracellular and (in the case of some members) extracellular activities. Systems, including knock-out animal models, should be now used with the aim of defining the correspondence between the in vitro regulatory role(s) attributed to individual members of this protein family and the in vivo function(s) of each S100 protein.

BACKGROUND: A principal goal of structure prediction is the elucidation of function. We have studied the ability of computed models to preserve the microenvironments of functional sites. In particular, 653 model structures of a calcium-binding protein (generated using an ab initio folding protocol) were analyzed, and the degree to which calcium-binding sites were recognizable was assessed. RESULTS: While some model structures preserve the calcium-binding microenvironments, many others, including some with low root mean square deviations (rmsds) from the crystal structure of the native protein, do not. There is a very weak correlation between the overall rmsd of a structure and the preservation of calcium-binding sites. Only when the quality of the model structure is high (rmsd less than 2 A for atoms in the 7 A local neighborhood around calcium) does the modeling of the binding sites become reliable. CONCLUSIONS: Protein structure prediction methods need to be assessed in terms of their preservation of functional sites. High-resolution structures are necessary for identifying binding sites such as calcium-binding sites.

A polymorphic dinucleotide (CA) sequence was isolated from a BAC clone containing the human 27-kD calbindin (CALB1) gene at 8q21. This polymorphism will be a useful genetic marker to study genetic variations of the CALB1 gene.

Determination of the metal-binding cooperativity of wild-type and mutant calbindin D9K by electrospray ionization mass spectrometry.

Rapid Commun Mass Spectrom. 1999; 13: 548-55

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Since the initial reports showing the ability of electrospray ionization mass spectrometry (ESI-MS) to study intact noncovalent biomolecular complexes, an increasing number of uses for this technique in studying biochemical systems is emerging. We have investigated the ability of ESI-MS to characterize the metal-binding properties of calcium (Ca2+) binding proteins by studying the incorporation of Ca2+ and cadmium (Cd2+) into wild-type and mutant calbindin D9K. ESI-MS showed that wild-type calbindin D9K binds two Ca2+ ions with similar affinities while the binding of two Cd2+ ions is sequential, as is the binding of the two Ca2+ or Cd2+ ions to the N56A mutant of calbindin. The binding of Ca2+ to the wild-type protein was clearly seen to be cooperative. These results demonstrate the potential efficacy of ESI-MS to discriminate between cooperative and independent site metal binding to metalloproteins.

Calbindin D28k is a protein abundant in the mammalian central nervous system and in epithelial tissue involved in Ca2+ transport. Human calbindin D28k was cloned into a Pet3a vector and expressed in Escherichia coli. The protein was purified in three steps: (i) heat precipitation of bacterial proteins, (ii) ion-exchange chromatography on a DEAE-cellulose column in the presence of calcium, and (iii) ion-exchange chromatography on a DEAE-Sephacel column in the presence of EDTA. The protein was then supplemented with calcium and dialyzed against neutral water. The final yield was 20-50 mg of pure, homogeneous calcium-loaded calbindin D28k per liter of bacterial culture. The identity and purity of the protein were confirmed by immunoblotting, SDS-polyacrylamide gel electrophoresis, and agarose gel electrophoresis in the absence and presence of calcium and 1H NMR spectroscopy. The entire expression and purification protocol takes only 3 days and is easy to scale up and down. It was designed to minimize degradation and deamidation.

Troponin I inhibitory peptide (96-115) has an extended conformation when bound to skeletal muscle troponin C.

Biochemistry. 1999; 38: 6911-7

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We have utilized CD and NMR spectroscopy to study the conformation of the troponin I (TnI) inhibitory peptide [TnI(96-115)] free in solution and when bound to troponin C (TnC). Analysis of the CD spectrum of the free peptide in aqueous solution indicates it is only approximately 3% helix. Upon complex formation with TnC, there is no change in total helix content compared to the sum of the free components. The NMR data support a predominantly extended conformation for the free peptide. TnI(96-115) bound to TnC was selectively observed by NMR using deuterated TnC (dTnC). For the 1:1 ratio of TnI(96-115) to dTnC used, 95% of the peptide was bound to dTnC. The chemical shifts of the TnC-bound peptide resonances are similar to those of the free peptide, indicating that the change in peptide conformation as a consequence of binding to TnC is small. For the TnC-bound TnI(96-115) peptide, the ratios of sequential Halpha-HN to intraresidue HN-Halpha NOE cross-peak volumes support a predominantly extended conformation, possibly kinked at Gly104. The results presented here are in agreement with sequence analysis predictions for TnI(96-115) as a free peptide or within the intact TnI sequence. The predominantly extended structure for the 96-115 inhibitory sequence segment of TnI with a kink at Gly104 may facilitate its binding alternately to actin or TnC in response to the Ca2+ signals that control thick and thin filament interactions during the contractile cycle.

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B(betabeta).

Protein Eng. 1999; 12: 395-405

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P26olf from olfactory tissue of frog, which may be involved in olfactory transduction or adaptation, is a Ca2+-binding protein with 217 amino acids. The p26olf molecule contains two homologous parts consisting of the N-terminal half with amino acids 1-109 and the C-terminal half with amino acids 110-217. Each half resembles S100 protein with about 100 amino acids and contains two helix-loop-helix Ca2+-binding structural motifs known as EF-hands: a normal EF-hand at the C-terminus and a pseudo EF-hand at the N-terminus. Multiple alignment of the two S100-like domains of p26olf with 18 S100 proteins indicated that the C-terminal putative EF-hand of each domain contains a four-residue insertion when compared with the typical EF-hand motifs in the S100 protein, while the N-terminal EF-hand is homologous to its pseudo EF-hand. We constructed a three-dimensional model of the p26olf molecule based on results of the multiple alignment and NMR structures of dimeric S100B(betabeta) in the Ca2+-free state. The predicted structure of the p26olf single polypeptide chain satisfactorily adopts a folding pattern remarkably similar to dimeric S100B(betabeta). Each domain of p26olf consists of a unicornate-type four-helix bundle and they interact with each other in an antiparallel manner forming an X-type four-helix bundle between the two domains. The two S100-like domains of p26olf are linked by a loop with no steric hindrance, suggesting that this loop might play an important role in the function of p26olf. The circular dichroism spectral data support the predicted structure of p26olf and indicate that Ca2+-dependent conformational changes occur. Since the C-terminal putative EF-hand of each domain fully keeps the helix-loop-helix motif having a longer Ca2+-binding loop, regardless of the four-residue insertion, we propose that it is a new, novel EF-hand, although it is unclear whether this EF-hand binds Ca2+. P26olf is a new member of the S100 protein family.

Intracellular neuronal calcium sensor proteins: a family of EF-hand calcium-binding proteins in search of a function.

Cell Tissue Res. 1999; 295: 1-12

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Intracellular neuronal calcium sensors (NCS) constitute a rapidly growing family of calcium-binding proteins which belong to the superfamily of EF-hand proteins. The NCS family includes as subgroups the recoverins and GCAPs (guanylyl cyclase-activating proteins), which are primarily expressed in retinal photoreceptor cells, and the frequenins and VILIPs (visinin-like proteins), which are widely but differentially expressed in the nervous system. In this review the recent developments in elucidating the functional activities of NCS proteins on signal transduction pathways in neurons are surveyed and discussed. We will focus our attention on calcium-dependent membrane association by the so-called calcium-myristoyl switch as a possible mechanism of signal transduction and on the roles of NCS proteins in intraneuronal signaling cascades, which are best studied in the visual and olfactory systems.

Analysis of the MRP8-MRP14 protein-protein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation.

J Biol Chem. 1999; 274: 183-8

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Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional "in vivo" approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.

High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins.

J Biomol NMR. 1999; 13: 233-47

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The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C, 15N-enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE-derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter-subunit NOEs (386) were identified from 13C-select, 13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 A). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca(2+)-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.

Energy-based de novo protein folding by conformational space annealing and an off-lattice united-residue force field: application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K.

Proc Natl Acad Sci U S A. 1999; 96: 2025-30

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The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.

Mutations in the N- and D-helices of the N-domain of troponin C affect the C-domain and regulatory function.

Biophys J. 1999; 76: 400-8

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Troponin C contains a 14-residue alpha-helix at the amino terminus, the N-helix, that calmodulin lacks. Deletion of the first 11-14 residues of troponin C alters function. In the present investigation a mutant lacking residues 1-7 of the N-helix has normal conformation, Ca2+ binding, and regulatory function. Thus, residues 8-14 of the N-helix are generally sufficient for troponin C function. In the x-ray structures of troponin C there is a salt bridge between Arg 11 in the N-helix and Glu 76 in the D-helix. Destroying the salt bridge by individually mutating the residues to Cys has no effect on function. However, mutation of both residues to Cys reduces troponin C's affinity for the troponin complex on the thin filament, reduces the stability of the N-domain in the absence of divalent cations, increases the Ca2+ affinity and reduces the cooperativity of the Ca2+Mg2+ sites in the C-domain, and alters the conformational change that takes place upon Ca2+ binding (but not Mg2+ binding) to the C-domain. Cross-linking with bis-(maleimidomethylether) partially restores function. The Ca2+-specific sites in the N-domain, those closest to the sites of the mutations, are unaffected in the assays employed. These results show that the N-helix is a critical structural element for interaction with and activation of the thin filament. Moreover, mutations in the N-helix affect the C-terminal domain, consistent with recent structural studies showing that the N-helix and C-terminal domain are physically close.

Procedures are described for significantly improving the sensitivity of the recently proposed TROSY (transverse relaxation-optimized spectroscopy) experiment (K. Pervushin et al., 1997, Proc. Natl. Acad. Sci. USA 94, 12366-12371). The TROSY experiment takes advantage of destructive interference between dipolar and chemical shift anisotropy relaxation mechanisms to achieve substantial reductions in resonance linewidths in heteronuclear correlation spectra; the effect is significant particularly for studies of large molecular weight systems at very high static magnetic field strengths. A (square root 2) improvement in the sensitivity of the TROSY experiment is achieved by implementation of the PEP (preservation of equivalent pathways) scheme (J. Cavanagh and M. Rance, 1990, J. Magn. Reson. 88, 72-85). An additional significant improvement in sensitivity for 15N-labeled samples in H2O solution is realized through a simple modification of the 1H-15N TROSY pulse sequence to return the water magnetization to its equilibrium position (+z axis) at the beginning of the acquisition period. Relaxation-induced imbalance between the coherence transfer pathways utilized in the TROSY refocusing period is shown theoretically and experimentally to give rise to additional unanticipated signals in TROSY spectra.

This review discusses the structure and properties of the isolated components of troponin, their interaction, and the mechanisms of regulation of contractile activity of skeletal and cardiac muscle. Data on the structure of troponin C in crystals and in solution are presented. The Ca2+-induced conformational changes of troponin C structure are described. The structure of troponin I is analyzed and its interaction with other components of actin filaments is discussed. Data on phosphorylation of troponin I by various protein kinases are presented. The role of troponin I phosphorylation in the regulation of contractile activity of the heart is analyzed. The structural properties of troponin T and its interaction with other components of thin filaments are described. Data on the phosphorylation of troponin T are presented and the effect of troponin T phosphorylation on contractile activity of different muscles is discussed. Modern models of the functioning of troponin are presented and analyzed.

Senescence marker protein-30 (SMP30), which we previously identified, is notable for its androgen-independent decrease in the livers of aging rats. Hepatocytes and renal tubular epithelia express large amounts of SMP30 in their cytosol throughout the tissue-maturing process and adulthood, but its level decreases thereafter. Upon cloning cDNAs that encode SMP30 in rats, mice, and humans, we found that the amino acid sequence of SMP30 is well conserved with remarkable homology among these species. However, this gene, which is so strongly conserved in these higher animals, does not appear in yeast. We also determined the genome organization and 5' flanking region of SMP30 in mouse genome. In the meantime, SMP30 turned out be identical to a Ca2+-binding protein called regucalcin (RC). To learn how this protein functions, we transfected Hep G2 cells with human SMP30 cDNA so that these cells stably express large amounts of SMP30. The results suggest that SMP30 regulates Ca2+ homeostasis by enhancing Ca2+-pumping activity in the plasma membranes. Thus, SMP30 seems to play a critical role in the highly differentiated functions of the liver and kidney and to exert a major impact on Ca2+ homeostasis. If so, down-regulation of SMP30 with aging would attribute greatly to the related deterioration of these organs, as indicated in this brief overview of the structure, expression, and function of SMP30.

A calcium binding protein from Entamoeba histolytica, (EhCaBP, M(r) approximately 15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (approximately 30%) with other known CaBPs. Almost complete sequence specific resonance assignments for (1)H, (13)C and (15)N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix-loop-helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the alpha-helices and beta-strands in EhCaBP resemble them.

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.

The interaction of troponin-C (TnC) with troponin-I (TnI) plays a central role in skeletal and cardiac muscle contraction. We have recently shown that the binding of Ca2+ to cardiac TnC (cTnC) does not induce an "opening" of the regulatory domain in order to interact with cTnI [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221; Spyracopoulos et al. (1997) Biochemistry 36, 12138-12146], which is in contrast to the regulatory N-domain of skeletal TnC (sTnC). This implies that the mode of interaction between cTnC and cTnI may be different than that between sTnC and sTnI. In sTnI, a region downstream from the inhibitory region (residues 115-131) has been shown to bind the exposed hydrophobic pocket of Ca2+-saturated sNTnC [McKay, R. T., et al. (1997) J. Biol. Chem. 272, 28494-28500]. The present study demonstrates that the corresponding region in cTnI (residues 147-163) binds to the regulatory domain of cTnC only in the Ca2+-saturated state to form a 1:1 complex, with an affinity approximately six times weaker than that between the skeletal counterparts. Thus, while Ca2+ does not cause opening, it is required for muscle regulation. The solution structure of the cNTnC.Ca2+.cTnI147-163 complex has been determined by multinuclear multidimensional NMR spectroscopy. The structure reveals an open conformation for cNTnC, similar to that of Ca2+-saturated sNTnC. The bound peptide adopts a alpha-helical conformation spanning residues 150-157. The C-terminus of the peptide is unstructured. The open conformation for Ca2+-saturated cNTnC in the presence of cTnI (residues 147-163) accommodates hydrophobic interactions between side chains of the peptide and side chains at the interface of A and B helices of cNTnC. Thus the mechanistic differences between the regulation of cardiac and skeletal muscle contraction can be understood in terms of different thermodynamics and kinetics equilibria between essentially the same structure states.

Calretinin (CR) is a 31.5 kDa EF-hand calcium binding protein. CR has a general neuronal localization and is a predominantly cytosolic protein. The biochemical properties of this protein are well characterized but its function is still unclear. It is often postulated that the presence of CR correlates with an increased survival ability of cells under pathological conditions connected with increased intracellular calcium levels. However, not all studies confirm such a relationship and they indicate that the presence of CR does not protect cells against calcium overload and that CR does not act as an intracellular calcium buffer. Recent results concerning CR function are discussed and the conclusion is proposed that CR acts as a calcium modulator rather than a calcium buffer.

Cloning and expression of a cDNA encoding a Vorticella convallaria spasmin: an EF-hand calcium-binding protein.

J Eukaryot Microbiol. 1999; 46: 165-73

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The stalked, ciliated protozoan Vorticella convallaria possesses a highly contractile cytoskeleton consisting of spasmonemes and myonemes. The major component of these contractile organelles is the calcium-binding protein(s) called spasmin. Cloning and characterization of spasmin would help elucidate this contractile system. Therefore, enriched spasmoneme protein preparations from these contractile stalks were used to produce a monoclonal antibody to spasmin. A monoclonal antibody, 1F5, was obtained that immunolocalized specifically to the spasmonemes and the myonemes and recognized a 20-kD calcium-binding protein in spasmoneme protein preparations. A putative spasmin cDNA was obtained from a V. convallaria cDNA library and the derived amino acid sequence of this cDNA revealed an acidic, 20-kD protein with calcium-binding helix-loop-helix domains. The physical properties of the putative spasmin were assessed by characterization of a recombinantly-produced spasmin protein. The recombinant spasmin protein was shown to bind calcium using calcium gel-shift assays and was recognized by the anti-spasmin antibody. Therefore, a V. convallaria spasmin was cloned and shown to be a member of the EF-hand superfamily of calcium-binding proteins.

Contractile activity of skeletal muscle is triggered by a Ca2+-induced "opening" of the regulatory N-domain of troponin C (apo-NTnC residues 1-90). This structural transition has become a paradigm for large-scale conformational changes that affect the interaction between proteins. The regulatory domain is comprised of two basic structural elements: one contributed by the N-, A-, and D-helices (NAD unit) and the other by the B- and C-helices (BC unit). The Ca2+-induced opening is characterized by a movement of the BC unit away from the NAD unit with a concomitant change in conformation at two hinges (Glu41 and Val65) of the BC unit. To examine the effect of low temperatures on this Ca2+-induced structural change and the implications for contractile regulation, we have examined nuclear magnetic resonance (NMR) spectral changes of apo-NTnC upon decreasing the temperature from 30 to 4 degrees C. In addition, we have determined the solution structure of apo-NTnC at 4 degrees C using multinuclear multidimensional NMR spectroscopy. Decreasing temperatures induce a decrease in the rates and amplitudes of pico to nanosecond time scale backbone dynamics and an increase in alpha-helical content for the terminal helices of apo-NTnC. In addition, chemical shift changes for the Halpha resonances of Val65 and Asp66, the hinge residues of the BC, unit were observed. Compared to the solution structure of apo-NTnC determined at 30 degrees C, the BC unit packs more tightly against the NAD unit in the solution structure determined at 4 degrees C. Concomitant with the tighter packing of the BC and NAD structural units, a decrease in the total exposed hydrophobic surface area is observed. The results have broad implications relative to structure determination of proteins in the presence of large domain movements, and help to elucidate the relevance of structures determined under different conditions of physical state and temperature, reflecting forces ranging from crystal packing to solution dynamics.

In order to elucidate if the inhibition mechanisms of Aluminum (Al) on intestinal calcium flux involve some possible action on calbindin-D9k, a series of in vivo and in vitro experiments were carried out in normal and in streptozotocin-induced diabetic male rats. The dose-response curves obtained from the in vitro studies indicate that, in the diabetic group (which has a lower content of calbindin-D9k), the effect of Al on JCa(ms) has a small dependence on rising Al concentration (0-10 microM). The parameters obtained from those curves: Emax (maximum reduction percentage of JCa(ms)) and ED50 (Al concentration that produces half of the highest inhibition) were significantly diminished in this group compared to control. Both s.c. injections of calcitriol (D3) at doses of 0.08 and 0.40 microg/kg body wt. per day and insulin (10 IU/kg body wt. per day), increase the inhibitory effect of Al to levels that did not differ from controls. In vivo gavage of 60 mg/kg body wt. per day of aluminum chloride for 1 week reveals that the degree of reduction of intestinal CaBP9k by Al is directly correlated to duodenal content of this protein (r2 = 0.683, P = 0.022).

Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.

Calcium binding to the class I alpha-1,2-mannosidase from Saccharomyces cerevisiae occurs outside the EF hand motif.

Biochemistry. 1999; 38: 1111-8

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Class I alpha-1,2-mannosidases are a family of Ca2+-dependent enzymes that have been conserved through eukaryotic evolution. These enzymes contain a conserved putative EF hand Ca2+-binding motif and nine invariant acidic residues. The catalytic domain of the alpha-1, 2-mannosidase from Saccharomyces cerevisiae was expressed in Pichia pastoris and was shown by atomic absorption and equilibrium dialysis to bind one Ca2+ ion with high affinity (KD = 4 x 10(-)7 M). Ca2+ protected the enzyme from thermal denaturation. Mutation of the 1st and 12th residues of the putative EF hand Ca2+ binding loop (D121N, D121A, E132Q, E132V, and D121A/E132V) had no effect on Ca2+ binding, demonstrating that the EF hand motif is not the site of Ca2+ binding. In contrast, three invariant acidic residue mutants (D275N, E279Q, and E438Q) lost the ability to bind 45Ca2+ following nondenaturing polyacrylamide gel electrophoresis whereas D86N, E132Q, E503Q, and E526Q mutants exhibited binding of 45Ca2+ similar to the wild-type enzyme. The wild-type enzyme had a Km and kcat of 0.5 mM and 12 s-1, respectively. The Km of E526Q was greatly increased to 4 mM with a small reduction in kcat to 5 s-1 whereas the kcat values of D86N and E132Q(V) were greatly reduced (0.005-0.007 s-1) with a decrease in Km (0.07-0.3 mM). The E503Q mutant is completely inactive. Asp275, Glu279, and Glu438 are therefore required for Ca2+ binding whereas Asp86, Glu132, and Glu503 are required for catalysis.

NMR investigation and secondary structure of domains I and II of rat brain calbindin D28k (1-93).

Eur J Biochem. 1999; 262: 933-8

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Calbindin D28k, a member of the troponin C superfamily of calcium-binding proteins, contains six putative EF hand domains but binds only four calcium-atoms: one at a binding site of very high affinity and three calcium-atoms at binding sites of lower affinity. The high-affinity site could be located within domain I while domains III, IV, and V bind calcium less tightly. The recombinant protein construct calb I-II (residues 1-93) comprising the first two EF hands affords a unique opportunity to study a pair of EF hands with one site binding calcium tightly and the second site empty. A series of heteronuclear 2D, 3D and 4D high-resolution NMR experiments were applied to calb I-II, and led to the complete assignment of the 1H, 13C and 15N resonances. The secondary structure of the protein was deduced from the size of the 3JHN-Halpha coupling constants, the chemical shift indices of 1Etaalpha, 13Calpha, 13C' and 13Cbeta nuclei and from an analysis of backbone NOEs observed in 3D and 4D NOESY spectra. Four major alpha-helices are identified: Ala13-Phe23, Gly33-Ala50, Leu54-Asp63, Val76-Leu90, while residues Ala2-Leu6 form a fifth, flexible helical segment. Two short beta-strands (Tyr30-Glu32, Lys72-Gly74) are found preceding helices B and D and are arranged in an anti-parallel interaction. Based on these data a structural model of calb I-II was constructed that shows that the construct adopts a tertiary structure related to other well-described calcium-binding proteins of the EF-hand family. Surprisingly, the protein forms a homodimer in solution, as was shown by its NMR characterization, size-exclusion chromatography and analytical ultra-centrifugation studies.

The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.

Effects of high pressure and temperature on the wild-type and F29W mutant forms of the N-domain of avian troponin C.

Biochim Biophys Acta. 1999; 1431: 53-63

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The N-domain of troponin C (residues 1-90) regulates muscle contraction through conformational changes induced by Ca2+ binding. A mutant form of the isolated domain of avian troponin C (F29W) has been used in previous studies to observe conformational changes that occur upon Ca2+ binding, and pressure and temperature changes. Here we set out to determine whether the point mutation itself has any effects on the protein structure and its stability to pressure and temperature in the absence of Ca2+. Molecular dynamics simulations of the wild-type and mutant protein structures suggested that both structures are identical except in the main chain and the loop I region near the mutation site. Also, the simulations proposed that an additional cavity had been created in the core of the mutant protein. To determine whether such a cavity would affect the behavior of the protein when subjected to high pressures and temperatures, we performed 1H-NMR experiments at 300, 400, and 500 MHz on the wild-type and F29W mutant forms of the chicken N-domain troponin C in the absence of Ca2+. We found that the mutant protein at 5 kbar pressures had a destabilized beta-sheet between the Ca2+-binding loops, an altered environment near Phe-26, and reduced local motions of Phe-26 and Phe-75 in the core of the protein, probably due to a higher compressibility of the mutant. Under the same pressure conditions, the wild-type domain exhibited little change. Furthermore, the hydrophobic core of the mutant protein denatured at temperatures above 47 degrees C, while the wild-type was resistant to denaturation up to 56 degrees C. This suggests that the partially exposed surface mutation (F29W) significantly destabilizes the N-domain of troponin C by altering the packing and dynamics of the hydrophobic core.

Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in the Zn2+-loaded and Zn2+-free states.

Biochemistry. 1999; 38: 1695-704

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The crystal structure of human psoriasin (S100A7) in the native, calcium-bound form has been determined from two crystal forms of the protein crystallized with and without divalent zinc. The overall structures of the dimeric protein closely resemble the previously determined holmium-substituted structure. The structures also reveal a zinc-binding site of the protein, which is formed by three histidines and an aspartate residue. Together, these residues coordinate the zinc ion in a way similar to the pattern seen in certain metalloproteases and in particular the collagenase family of proteins. Sequence comparison suggests that this zinc site is present in a number of the remaining members of the S100 family. The structure of S100A7 crystallized in the absence of zinc further shows that loss of zinc results in a reorganization of the adjacent empty and distorted EF-hand loop, causing it to resemble a calcium-loaded EF-hand.

Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I.

Biochemistry. 1998; 37: 12419-30

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The structure of the regulatory domain of chicken skeletal troponin-C (residues 1-90) when complexed with the major inhibitory region (residues 96-148) of chicken skeletal troponin-I was determined using multinuclear, multidimensional NMR spectroscopy. This complex represents the first interaction formed between the regulatory domain of troponin-C and troponin-I after calcium binding in the regulation of muscle contraction. The stoichiometry of the complex was determined to be 1:1, with a dissociation constant in the 1-40 microM range. The structure of troponin-C in the complex was calculated from 1039 NMR distance and 111 dihedral angle restraints. When compared to the structure of this domain in the calcium saturated "open" form but in the absence of troponin-I, the bound structure appears to be slightly more "closed". The troponin-I peptide-binding site was found to be in the hydrophobic pocket of calcium saturated troponin-C, using edited/filtered NMR experiments and chemical shift mapping of changes induced in the regulatory domain upon peptide binding. The troponin-I peptide (residues 96-148) was found to bind to the regulatory domain of troponin-C very similarly, but not identically, to a shorter troponin-I peptide (region 115-131) thought to represent the major interaction site of troponin-I for this domain of troponin-C.

When size is important. Accommodation of magnesium in a calcium binding regulatory domain.

J Biol Chem. 1998; 273: 28994-9001

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The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide 1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (koff = 380 +/- 40 s-1 compared with approximately 10,000 s-1 at 25 degreesC). The Mg2+-saturated loop II undergoes conformational exchange on the 100-microseconds time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.

Molecular cloning and expression of active Ole e 3, a major allergen from olive-tree pollen and member of a novel family of Ca2+-binding proteins (polcalcins) involved in allergy.

Eur J Biochem. 1998; 258: 454-9

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A cDNA encoding Ole e 3, a major allergen from olive-tree pollen, has been cloned and sequenced. A strategy based on two-step PCR amplification towards the 5' end and 3' end, with an internal specific primer, has been used. The isolated cDNA contains an open reading frame coding for a polypeptide of 84 amino acids, which is in agreement with the composition and molecular mass of the natural allergen, exhibiting two 12-residue segments homologous to Ca2+-binding sites of EF-hand type. The cDNA was inserted into the pET-11b expression vector and over-expressed in Escherichia coli. The purified recombinant protein shows identical secondary structure to that of the natural allergen and is able to bind both IgE from sera of patients allergic to olive pollen and polyclonal antibodies raised against olive-pollen Ole e 3. The capacity of binding Ca2+ has been demonstrated for both natural and recombinant allergens. RNA transcripts of Ole e 3 were only detected in pollen tissue. Northern-blot and Western-blot analyses of poly(A)+ RNA and protein extracts, respectively, obtained from a variety of olive-tree-related and nonrelated mature pollens demonstrated the presence of Ole e 3 homologous proteins. This indicates a sequence conservation and widespread distribution for this family of Ca2+-binding proteins that can be responsible for allergenic cross-reactivity. We suggest the tentative generic name of polcalcins for the members of this family of Ca2+-binding proteins from pollen.

S100 proteins are a family of dimeric calcium-binding proteins implicated in several cancers and neurological diseases. Calbindin D9k is an unusual monomeric member of the S100 family. A calbindin D9k mutant containing a novel calcium-induced helix is characterized. Based on sequence comparison, this helix could be a component of other S100 proteins and a factor in target protein binding. The origin of structural differences between three reported apo S100 dimer structures is verified. We conclude that the differences are a result of modeling rather than a function of different target binding properties. A mechanism for target protein binding is suggested.

Sequence and context dependence of EF-hand loop dynamics. An 15N relaxation study of a calcium-binding site mutant of calbindin D9k.

Biochemistry. 1998; 37: 2586-95

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The influence of amino acid sequence and structural context on the backbone dynamics of EF-hand calcium-binding loops was investigated using 15N spin relaxation measurements on the calcium-free state of the calbindin D9k mutant (A14D+A15Delta+P20Delta+N21G+P43M), in which the N-terminal pseudo-EF-hand loop, characteristic of S100 proteins, was engineered so as to conform with the C-terminal consensus EF-hand loop. The results were compared to a previous study of the apo state of the wild-type-like P43G calbindin D9k mutant. In the helical regions, the agreement with the P43G data is excellent, indicating that the structure and dynamics of the protein core are unaffected by the substitutions in the N-terminal loop. In the calcium-binding loops, the flexibility is drastically decreased compared to P43G, with the modified N-terminal loop showing a motional restriction comparable to that of the surrounding helixes. As in P43G, the motions in the C-terminal loop are less restricted than in the N-terminal loop. Differences in key hydrogen-bonding interactions correlate well with differences in dynamics and offer insights into the relationship between structure and dynamics of these EF-hand loops. It appears that the entire N-terminal EF-hand is built to form a rigid structure that allows calcium binding with only minor rearrangements and that the structural and dynamical properties of the entire EF-hand--rather than the loop sequence per se--is the major determinant of loop flexibility in this system.

Forty-five distinct subfamilies of EF-hand proteins have been identified. They contain from two to eight EF-hands that are recognizable by amino acid sequence as being statistically similar to other EF-hand domains. All proteins within one subfamily are congruent to one another, i.e. the dendrogram computed from one of the EF-hand domains is similar, within statistical error, to the dendrogram computed from another(s) domain. Thirteen subfamilies--including Calmodulin, Troponin C, Essential light chain, Regulatory light chain--referred to collectively as CTER, are congruent with one another. They appear to have evolved from a single ur-domain by two cycles of gene duplication and fusion. The subfamilies of CTER subsequently evolved by gene duplications and speciations. The remaining 32 subfamilies do not show such general patterns of congruence; however, some--such as S100, intestinal calcium binding protein (calbindin 9 kd), and trichohylin--do not form congruent clusters of subfamilies. Nearly all of the domains 1, 3, 5, and 7 are most similar to other ODD domains. Correspondingly the EVEN numbered domains of all 45 subfamilies most closely resemble EVEN domains of other subfamilies. Many sequence and chemical characteristics do not show systemic trends by subfamily or species of host organisms; such homoplasy is widespread. Eighteen of the subfamilies are heterochimeric; in addition to multiple EF-hands they contain domains of other evolutionary origins.

Desulfovibrio vulgaris cytochrome c3 is a 14 kDa tetrahaem cytochrome that plays a central role in energy transduction. The three-dimensional structure of the ferrocytochrome at pH 8.5 was solved through two-dimensional 1H-NMR. The structures were calculated using a large amount of experimental information, which includes upper and lower distance limits as well as dihedral angle restraints. The analysis allows for fast-flipping aromatic residues and flexibility in the haem plane. The structure was determined using 2289 upper and 2390 lower distance limits, 63 restricted ranges for the phi torsion angle, 88 stereospecific assignments out of the 118 stereopairs with non-degenerate chemical shifts (74.6%), and 115 out of the 184 nuclear Overhauser effects to fast-flipping aromatic residues (62.5%), which were pseudo-stereospecifically assigned to one or the other side of the ring. The calculated NMR structures are very well defined, with an average root-mean-square deviation value relative to the mean coordinates of 0.35 A for the backbone atoms and 0.70 A for all heavy-atoms. Comparison of the NMR structures of the ferrocytochrome at pH 8.5 with the available X-ray structure of the ferricytochrome at pH 5.5 reveals that the general fold of the molecule is very similar, but that there are some distinct differences. Calculation of ring current shifts for the residues with significantly different conformations confirms that the NMR structures represent better its solution structure in the reduced form. Some of the localised differences, such as a reorientation of Thr24, are thought to be state-dependent changes that involve alterations in hydrogen bond networks. An important rearrangement in the vicinity of the propionate groups of haem I and involving the covalent linkage of haem II suggests that this is the critical region for the functional cooperativities of this protein.

Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding but not for dimerization.

Cell Calcium. 1998; 24: 137-51

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S100 proteins are a family of small dimeric proteins characterized by two EF hand type Ca2+ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases S100 proteins are thought to function by binding to, and thereby regulating, cellular target proteins in a Ca2+ dependent manner. To describe for one member of the family, S100A1, structural requirements underlying target protein binding, we generated specifically mutated S100A1 derivatives and characterized their interaction with the alpha subunit of the actin capping protein CapZ shown here to represent a direct binding partner for S100A1. Chemical cross-linking, ligand blotting and fluorescence emission spectroscopy reveal that removal of, or mutations within, the sequence encompassing residues 88-90 in the unique C-terminal region of S100A1 interfere with binding to CapZ alpha and to TRTK-12, a synthetic CapZ alpha peptide. The S100A1 sequence identified contains a cluster of three hydrophobic residues (Phe-88, Phe-89 and Trp-90) at least one of which--as revealed by qualitative phenyl Sepharose binding and hydrophobic fluorescent probe spectroscopy--is exposed on the protein surface of Ca2+ bound S100A1. As homologous hydrophobic residues in the closely related S100B protein were shown by NMR spectroscopy of Ca(2+)-free S100B dimers to provide intersubunit contacts [Kilby P.M., van Eldik L.J., Roberts G.C.K. The solution structure of the bovine S100B dimer in the calcium-free state. Structure 1996; 4: 1041-1052; Drohat A.C., Amburgey J.C., Abildgaard F., Starich M.R., Baldisseri D., Weber D.J. Solution structure of rat apo-S100B (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35: 11,577-11,588], we characterized the physical state of the various S100A1 derivatives. Analytical gel filtration and chemical cross-linking show that dimer formation is not compromised in S100A1 mutants lacking residues 88-90 or containing specific amino acid substitutions in this sequence. Thus a cluster of hydrophobic residues in the C-terminal region of S100A1 is essential for target protein binding but dispensable for dimerization, a situation possibly met in other S100 proteins as well.

Molecular dynamics study of calbindin D9k in the apo and singly and doubly calcium-loaded states.

Proteins. 1998; 33: 265-84

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Molecular dynamics simulations based on detailed atomic models are used to examine the structure and dynamics of calbindin D9k, a protein possessing a pair of EF-hands able to bind two calcium ions in a cooperative fashion. Trajectories for the apo and singly (in the C-terminal binding site) and doubly loaded structures are generated and analyzed. Each system is solvated in a 27 A radius sphere of 2,285 explicit water molecules. The influence of the remaining bulk is incorporated through a stochastic boundary potential including a solvent reaction field. Long-range electrostatic interactions are treated with a special method and are not truncated. The average structural and dynamic properties upon calcium binding are studied at the atomic level to gain insight into the cooperative interactions between the two binding sites. Results from the trajectories are compared with data from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. NMR 15N and 13C(alpha) backbone relaxation order parameters and crystallographic B-factors are calculated. Generally, there is a good qualitative agreement between calculated and observed properties. Results confirm that the doubly loaded state is closer, both structurally and dynamically, to the singly loaded state than either of these is to the apo state. It is observed that both hydrogen bonding and the packing of nonpolar side chains contribute to the coupling between the calcium binding sites. Two backbone-to-backbone hydrogen bonds linking the calcium-binding EF-hands (Leu23-0 ... HN-Val61 and Val61-O ... HN-Leu23) are sensitive to the state of occupancy. Residues Leu23 and Val61 exhibit the smallest rms fluctuations of the entire protein in the D state. In addition, the van der Waals interaction of Val61 with the rest of the protein varies with the calcium-binding state.

Lipoxygenases catalyze the biosynthesis of leukotrienes, lipoxins, and other lipid-derived mediators that are involved in a wide variety of pathophysiological processes, including inflammation, allergy, and tumorigenesis. Mammalian lipoxygenases are activated by a calcium-mediated translocation to intracellular membranes upon cell stimulation, and cooperate with cytosolic phospholipase A2 at the membrane surface to generate eicosanoids. Although it has been documented that plant cell stimulation increases intracellular Ca2+ concentration and activates cytosolic phospholipase A2, followed by lipoxygenase-catalyzed conversion of the liberated linolenic acid to jasmonic acid, no evidence is available for Ca2+-regulated membrane binding and activity of plant lipoxygenases. Plant lipoxygenases, unlike their mammalian counterparts, are believed to function independently of calcium or membranes. Here we present spectroscopic evidence for a calcium-regulated membrane-binding mechanism of soybean lipoxygenase-1 (L-1). Both calcium and membrane binding affect the structure and the mode of action of L-1. Free L-1 in solution is less accessible to the polar solvent and converts linoleic acid to conjugated dienes, whereas surface binding increases solvent accessibility and stimulates conjugated ketodiene production. Calcium exerts a biphasic effect on the structure and activity of L-1. Our results uncover a new regulatory mechanism for plant lipoxygenases and delineate common features in animal and plant cell signaling pathways.

Cloning of a cDNA encoding a new calcium-binding protein from Dictyostelium discoideum and its developmental regulation.

FEBS Lett. 1998; 441: 302-6

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By employing 2D-PAGE, a protein differentially expressed during the development of Dictyostelium discoideum was discovered. The full cDNA of this protein was cloned using RT-PCR. The deduced protein is composed of 166 amino acid residues containing four EF-hand domains typical for calcium-binding proteins and was named CBP3. This protein shows little amino acid sequence homology with the other calcium-binding proteins from D. discoideum except EF-hand domains. The CBP3 mRNA was absent in vegetative amebas and accumulated maximally at 6 h of the development on filters. The mRNA level decreased thereafter and disappeared after 12 h of the development, while the protein level peaked at 8 h of development and remained constant thereafter. The mobility of CBP3 on SDS gel was shifted by treatment with EGTA, confirming the Ca2+-binding activity of the protein.

We have used fluorescence resonance energy transfer to investigate the conformation of the apo and calcium-loaded states of the regulatory N-terminal domain of full-length troponin C mutants from skeletal muscle. The mutants studied each contained a single tryptophan residue (position 22 or 90) and a single cysteine residue (position 52 or 101). The intrinsic fluorophore in each mutant served as an energy donor and the cysteine was conjugated to the acceptor probe 5-(iodoacetamidoethyl)amino-naphthalene-1-sulfonic acid. The distributions of two intersite distances (between residues 22 and 52, and residues 90 and 52) were broad in the apo state, indicative of considerable structural dynamics. These distributions were shifted to longer distances and considerably sharpened in the calcium-loaded state. The shifts to longer distances by 8 to 11 A indicate a calcium-induced opening of the N-terminal domain conformation. The transition of the troponin C structure from a closed conformation to an open conformation is accompanied by a substantial reduction of structural fluctuations that dominate in the apo structure as evidenced from the large decrease of the widths of the distributions. This highly constrained open conformation is required as part of the structural basis to facilitate productive interaction between troponin C and troponin I to trigger contraction in skeletal muscle.

The three-dimensional structure of calcium-loaded regulatory, i.e. N-terminal, domain (1-91) of human cardiac troponin C (cNTnC) was determined by NMR in water/trifluoroethanol (91:9 v/v) solution. The single-calcium-loaded cardiac regulatory domain is in a "closed" conformation with comparatively little exposed hydrophobic surface. Difference distance matrices computed from the families of Ca2+-cNTnC, the apo and two-calcium forms of the skeletal TnC (sNTnC) structures reveal similar relative orientations for the N, A, and D helices. The B and C helices are closer to the NAD framework in Ca2+-cNTnC and in apo-sNTnC than in 2.Ca2+-sNTnC. However, there is an indication of a conformational exchange based on broad 15N resonances for several amino acids measured at several temperatures. A majority of the amides in the alpha-helices and in the calcium binding loop exhibit very fast motions with comparatively small amplitudes according to the Lipari-Szabo model. A few residues at the N and C termini are flexible. Data were recorded from nonlabeled and 15N-labeled samples, and backbone dynamics was investigated by 15N T1, T2, and heteronuclear nuclear Overhauser effect as well as by relaxation interference measurements.

Hydrophobic core residues have a marked influence on the Ca2+-binding properties of calbindin D9k, even though there are no direct contacts between these residues and the bound Ca2+ ions. Eleven different mutants with substitutions in the hydrophobic core were produced, and their equilibrium Ca2+-binding constants measured from Ca2+ titrations in the presence of chromophoric chelators. The Ca2+-dissociation rate constants were estimated from Ca2+ titrations followed by 1H NMR1 and were measured more accurately using stopped-flow fluorescence. The parameters were measured at four KCl concentrations to assess the salt dependence of the perturbations. The high similarity between the NMR spectra of mutants and wild-type calbindin D9k suggests that the structure is largely unperturbed by the substitutions. More detailed NMR investigations of the mutant in which Val61 is substituted by Ala showed that the mutation causes only very minimal perturbations in the immediate vicinity of residue 61. Substitutions of alanines or glycines for bulky residues in the center of the core were found to have significant effects on both Ca2+ affinity and dissociation rates. These substitutions caused a reduction in affinity and an increase in off-rate. Small effects, both increases and decreases, were observed for substitutions involving residues far from the Ca2+ sites and toward the outer part of the hydrophobic core. The mutant with the substitution Phe66 --> Trp behaved differently from all other mutants, and displayed a 25-fold increase in overall affinity of binding two Ca2+ ions and a 6-fold reduction in calcium dissociation rate. A strong correlation (R = 0.94) was found between the observed Ca2+-dissociation rates and affinities, as well as between the salt dependence of the off-rate and the distance to the nearest Ca2+-coordinating atom. There was also a strong correlation (R = 0.95) between the Ca2+ affinity and stability of the Ca2+ state and a correlation (R = 0. 69) between the Ca2+ affinity and stability of the apo state, as calculated from the results in the present and preceding paper in this issue [Julenius, K., Thulin, E., Linse, S., and Finn, B. E. (1998) Biochemistry 37, 8915-8925]. The change in salt dependencies of koff and cooperativity were most pronounced for residues completely buried in the core of the protein (solvent accessible surface area approximately 0). Altogether, the results suggest that the hydrophobic core residues promote Ca2+ binding both by contributing to the preformation of the Ca2+ sites in the apo state and by preferentially stabilizing the Ca2+-bound state.

The three-dimensional structure of Ca(2+)-bound calcyclin: implications for Ca(2+)-signal transduction by S100 proteins.

Structure. 1998; 6: 223-31

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BACKGROUND: Calcyclin is a member of the S100 subfamily of EF-hand Ca(2+)-binding proteins. This protein has implied roles in the regulation of cell growth and division, exhibits deregulated expression in association with cell transformation, and is found in high abundance in certain breast cancer cell lines. The novel homodimeric structural motif first identified for apo calcyclin raised the possibility that S100 proteins recognize their targets in a manner that is distinctly different from that of the prototypical EF-hand Ca2+ sensor, calmodulin. The NMR solution structure of Ca(2+)-bound calcyclin has been determined in order to identify Ca(2+)-induced structural changes and to obtain insights into the mechanism of Ca(2+)-triggered target protein recognition. RESULTS: The three-dimensional structure of Ca(2+)-bound calcyclin was calculated with 1372 experimental constraints, and is represented by an ensemble of 20 structures that have a backbone root mean square deviation of 1.9 A for the eight helices. Ca(2+)-bound calcyclin has the same symmetric homodimeric fold as observed for the apo protein. The helical packing within the globular domains and the subunit interface also change little upon Ca2+ binding. A distinct homology was found between the Ca(2+)-bound states of the calcyclin subunit and the monomeric S100 protein calbindin D9k. CONCLUSIONS: Only very modest Ca(2+)-induced changes are observed in the structure of calcyclin, in sharp contrast to the domain-opening that occurs in calmodulin and related Ca(2+)-sensor proteins. Thus, calcyclin, and by inference other members of the S100 family, must have a different mode for transducing Ca2+ signals and recognizing target proteins. This proposal raises significant questions concerning the purported roles of S100 proteins as Ca2+ sensors.

A novel calcium-sensitive switch revealed by the structure of human S100B in the calcium-bound form.

Structure. 1998; 6: 211-22

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BACKGROUND: S100B is a homodimeric member of the EF-hand calcium-binding protein superfamily. The protein has been implicated in cellular processes such as cell differentiation and growth, plays a role in cytoskeletal structure and function, and may have a role in neuropathological diseases, such as Alzheimers. The effects of S100B are mediated via its interaction with target proteins. While several studies have suggested that this interaction is propagated through a calcium-induced conformational change, leading to the exposure of a hydrophobic region of S100B, the molecular details behind this structural alteration remain unclear. RESULTS: The solution structure of calcium-saturated human S100B (Ca(2+)-S100B) has been determined by heteronuclear NMR spectroscopy. Ca(2+)-S100B forms a well defined globular structure comprising four EF-hand calcium-binding sites and an extensive hydrophobic dimer interface. A comparison of Ca(2+)-S100B with apo S100B and Ca(2+)-calbindin D9k indicates that while calcium-binding to S100B results in little change in the site I EF-hand, it induces a backbone reorientation of the N terminus of the site II EF-hand. This reorientation leads to a dramatic change in the position of helix III relative to the other helices. CONCLUSIONS: The calcium-induced reorientation of calcium-binding site II results in the increased exposure of several hydrophobic residues in helix IV and the linker region. While following the general mechanism of calcium modulatory proteins, whereby a hydrophobic target site is exposed, the 'calcium switch' observed in S100B appears to be unique from that of other EF-hand proteins and may provide insights into target specificity among calcium modulatory proteins.

Transcriptional regulation of rat calbindin expression during development determined by bacterially expressed protein.

J Nutr Sci Vitaminol (Tokyo). 1998; 44: 137-49

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Calbindin-D9k expression in intestinal mucosal cells reveals a specific pattern during development in rats. It shows a low basal level in suckling and adult rats, but after weaning at 21 d of age, increases to three times that of the basal level for several days only, around 24 d. We attempted to clarify whether the regulation of developmental change was at the transcriptional or post-transcriptional level. The calbindin-D9k protein and mRNA concentrations during pre- and postweaned development were determined by Western blot and Northern blot analysis, respectively, and compared with calcium binding activity by 45Ca. For Western blot analysis, a corresponding antibody was raised in rabbit using a bacterially expressed fusion protein, glutathione S-transferase (GST, EC 2.5.1.18), and calbindin-D9k. Calbindin-D9k cDNA was linked to a GST gene within a molecule of vector plasmid and a fusion protein was expressed in Escherichia coli. There were significant (p < 0.001) correlations between calbindin-D9k protein, mRNA concentrations and calcium-binding activity: r = 0.90 for protein vs. mRNA, r = 0.93 for protein vs. binding activity and r = 0.95 for mRNA vs. binding activity. These results indicate that calbindin-D9k expression during postnatal development is regulated at the transcriptional level.

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

Protein Sci. 1998; 7: 983-93

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The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.

Calcium-induced exposure of a hydrophobic surface of mouse ALG-2, which is a member of the penta-EF-hand protein family.

J Biochem (Tokyo). 1998; 124: 1170-7

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ALG-2 is a 22 kDa EF-hand type Ca2+-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca2+-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca2+-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca2+. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca2+-concentration dependent manner, the half-maximal effect occurring at approximately 6 microM. Mg2+ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.

Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3: spectroscopic investigations of its transition metal ion complexes, and three-dimensional structural model.

Biochim Biophys Acta. 1998; 1448: 264-76

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A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.

The NMR solution structure of human glutaredoxin in the fully reduced form.

J Mol Biol. 1998; 280: 687-701

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The determination of the nuclear magnetic resonance (NMR) solution structure of fully reduced human glutaredoxin is described. A total of 1159 useful nuclear Overhauser effect (NOE) upper distance constraints and 187 dihedral angle constraints were obtained as the input for the structure calculations for which the torsion angle dynamics program DYANA has been utilized followed by energy minimization in water with the AMBER force field as implemented in the program OPAL. The resulting 20 conformers have an average root-mean-square deviation value relative to the mean coordinates of 0.54 A for all the backbone atoms N, Calpha and C', and of 1.01 A for all heavy atoms. Human glutaredoxin consists of a four-stranded mixed beta-sheet composed of residues 15 to 19, 43 to 47, 72 to 75 and 78 to 81, and five alpha-helices composed of residues 4 to 9, 24 to 34, 54 to 65, 83 to 91, and 94 to 100. Comparisons with the structures of Escherichia coli glutaredoxin-1, pig liver glutaredoxin and human thioredoxin were made. Electrostatic calculations on the human glutaredoxin structure and that of related proteins provide an understanding of the variation of pKa values for the nucleophilic cysteine in the active site observed among these proteins. In addition, the high-resolution NMR solution structure of human glutaredoxin has been used to model the binding site for glutathione and for ribonucleotide reductase B1 by molecular dynamics simulations.

Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes. Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium. Using aequorin, it is shown that B. subtilis cells tightly regulate intracellular Ca2+ levels. This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling. Also, B. subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium. Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect. In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+. Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B. subtilis, four putative Ca2+ binding proteins were found, including AhpC. Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity.

J Mol Biol. 1998; 278: 667-86

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The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample.Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively.In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.

S100A1 utilizes different mechanisms for interacting with calcium-dependent and calcium-independent target proteins.

Biochemistry. 1998; 37: 17429-38

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While previous studies have identified target proteins that interact with S100A1 in a calcium-dependent manner as well as target proteins that interact in a calcium-independent manner, the molecular mechanisms of S100A1-target protein interaction have not been elucidated. In this study, point and deletion mutants of S100A1 were used to investigate the contribution of carboxyl terminal amino acids to S100A1 interaction with calcium-dependent and calcium-independent target proteins. First, a recombinant rat S100A1 protein (recS100A1) expressed in bacteria exhibited physical and chemical properties indistinguishable from native S100A1. Next, proteins lacking the carboxyl-terminal nine residues of recS100A1 (Delta85-93), or containing alanine substitutions at Phe 88 (F88A), Phe 89 (F89A), or Trp 90 (W90A), both Phe 88 and Phe 89 (F88/89A), or all three aromatic residues (F88/89A-W90A) were recombinantly expressed. Like recS100A1, F88A, F89A, and W90A proteins interacted with phenyl-Sepharose in a calcium-dependent manner. However, the Delta85-93 protein did not interact with phenyl-Sepharose, indicating that a phenyl-Sepharose-binding region (PSBR) of recS100A1 had been disrupted. The F88/89A and F88/89A-W90A proteins exhibited reduced calcium-dependent interaction with phenyl-Sepharose when compared with recS100A1, demonstrating that the carboxyl-terminal aromatic residues Phe 88, Phe 89, and Trp 90 comprise the PSBR of S100A1. Fluorescence studies showed that the Delta85-93 protein exhibited reduced calcium-dependent interaction with the dodecyl CapZ peptide, TRTK, while W90A bound TRTK with a Kd of 5.55 microM. These results demonstrate that the calcium-dependent target protein-binding site and the PSBR are indistinguishable. In contrast to the calcium-dependent target TRTK, activation of the calcium-independent target protein aldolase A by the point and deletion mutant S100A1s was indistinguishable from native S100A1. These results demonstrate that carboxyl-terminal residues are not required for S100A1 modulation of calcium-independent target protein aldolase A. Alltogether, these results indicate that S100A1 utilizes distinct mechanisms for interaction with calcium-independent and calcium-dependent target proteins.

The effect of 1,25-vitamin D3 on calbindin-D and calcium-metabolic variables in the rat.

Pharmacol Toxicol. 1998; 82: 118-21

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Intraperitoneal injection of 1,25-(OH)2D3 4 micrograms/kg was given to 84 calcium- and vitamin D-repleted Wistar rats and samples of plasma, duodenal mucosa and renal tissue were taken after 0, 3, 6, 12, 24, 48 and 96 hr (n = 12 at each time interval). Plasma-ionized Ca increased after 6 hr, reached a maximum after 24 hr and returned to the initial values after 96 hr. The concentrations of renal calbindin-D28k and intestinal calbindin-D9k did not increase until 48 hr after injection and remained elevated until 96 hr after. Therefore, significantly elevated concentrations of the cytosolic calbindin-D were found at a time with normal values of plasma Ca. The present data suggest that calbindin-D does not alone increase the transcellular Ca transport and, therefore, supports the view that calbindin-Ds may serve as Ca buffer proteins.

Novel insights into structure and function of MRP8 (S100A8) and MRP14 (S100A9).

Biochim Biophys Acta. 1998; 1448: 200-11

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The two migration inhibitory factor- (MIF)-related protein-8 (MRP8; S100A8) and MRP14 (S100A9) are two calcium-binding proteins of the S100 family. These proteins are expressed during myeloid differentiation, are abundant in granulocytes and monocytes, and form a heterodimeric complex in a Ca2+-dependent manner. Phagocytes expressing MRP8 and MRP14 belong to the early infiltrating cells and dominate acute inflammatory lesions. In addition, elevated serum levels of MRP8 and MRP14 have been found in patients suffering from a number of inflammatory disorders including cystic fibrosis, rheumatoid arthritis, and chronic bronchitis, suggesting conceivable extracellular roles for these proteins. Although a number of possible functions for MRP8/14 have been proposed, the biological function still remains unclear. This review addresses recent developments regarding the MRP14-mediated promotion of leukocyte-endothelial cell-interactions and the characterization of MRP8/14 heterodimers as a fatty acid binding protein complex. In view of the current knowledge, the authors will hypothesize that MRP8 and MRP14 play an important role in leukocyte trafficking, but do not affect neutrophil effector functions.

Induction of a spectroscopically defined transition by guanidinium hydrochloride on a recombinant calcium binding protein from Entamoeba histolytica.

FEBS Lett. 1998; 441: 71-6

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Sequence analysis and metal ion binding studies reported earlier have established that the calcium binding protein (CaBP) from the parasitic ameboid Entamoeba histolytica protein has four canonical EF hand motifs which bind calcium. Equilibrium denaturation studies on both the apo and the holo forms of this protein indicate the presence of stable transition intermediates at low denaturant concentrations as revealed by the binding of the non-specific hydrophobic dye ANS. Fast reaction kinetics shows that the binding of the Gdn+ ions at or near the Ca2+ sites in the N-terminal domain influences metal ion binding to the sites in the C-terminal domain. Isothermal calorimetric titrations performed using low GdnHCl concentrations reveal the presence of two binding sites of low affinity, both being endothermic in nature. Thus the stabilization of CaBP observed at low GdnHCl concentration represents a native-like intermediate, with the Gdn+ ions mimicking Ca2+ binding at the N-terminal domain of this protein.

BACKGROUND: The S100 family consists of small acidic proteins, belonging to the EF-hand class of calcium-binding proteins. They are primarily regulatory proteins, involved in cell growth, cell structure regulation and signal transduction. Psoriasin (S100A7) is an 11.7 kDa protein that is highly upregulated in the epidermis of patients suffering from the chronic skin disease psoriasis. Although its exact function is not known, psoriasin is believed to participate in the biochemical response which follows transient changes in the cellular Ca2+ concentration. RESULTS: The three-dimensional structure of holmium-substituted psoriasin has been determined by multiple anomalous wavelength dispersion (MAD) phasing and refined to atomic resolution (1.05 A). The structure represents the most accurately determined structure of a calcium-binding protein. Although the overall structure of psoriasin is similar to those of other S100 proteins, several important differences exist, mainly in the N-terminal EF-hand motif that contains a distorted loop and lacks a crucial calcium-binding residue. It is these minor differences that may account for the different specificities among members of this family. CONCLUSIONS: The structure of human psoriasin reveals that this protein, in contrast to other S100 proteins with known structure, is not likely to strongly bind more than one calcium ion per monomer. The present study contradicts the idea that calcium binding induces large changes in conformation, as suggested by previously determined structures of apo forms of S100 proteins. The substitution of Ca2+ ions in EF-hands by lanthanide ions may provide a general vehicle for structure determination of S100 proteins by means of MAD phasing.

This study focuses on a closed net of electron-pair donor-acceptor interactions, present in the core of all metal-bound EF-hand pairs, that link both metal ions across a short two-stranded beta-sheet. A molecular model based on the above cycle of interactions was studied using semi-empirical molecular orbital quantum mechanical methods. The calculations indicate that the interactions in the model cycle are cooperative, that is, that the interaction energy of the cyclic structure is greater than that of the sum of isolated interactions between its components. The cooperativity in this cycle can be attributed to an increase in the stability of the interactions resulting from a mutual polarisation of the associated groups. The predicted polarisation of the amide groups in the cycle is in agreement with experimental NMR 15N deshielding observed for these amide groups upon metal binding. Experimental observations of strengthening of the beta-sheet hydrogen bonds are also consistent with the model calculations. By this mechanism, the binding of the first metal ion would enhance the binding of the second metal ion, and thus, the intradomain cooperativity in cation binding of calmodulin and related EF-hand proteins can be ascribed, at least partly, to this short-range molecular mechanism.

Antibodies reactive with S100 protein are useful markers in a diagnostic immunohistochemistry laboratory dealing with cutaneous tumors. However, S100 protein is not a single protein but instead a group of S100 proteins with diverse functions. S100 proteins constitute a family of acidic calcium-binding proteins that are important in intracellular calcium metabolism. Recent evidence that some S100 proteins are secreted makes it likely that they are also involved in cell-cell interactions. The exploration of the status of the different members of the S100 family may yield not only diagnostic clues but also relevant functional information about the cells. Considerable recent progress has been made in our understanding of S100 proteins. This review surveys some of these findings that may be either directly or indirectly relevant to cutaneous pathology.

Calcium sensor proteins translate transient increases in intracellular calcium levels into metabolic or mechanical responses, by undergoing dramatic conformational changes upon Ca2+ binding. A detailed analysis of the calcium binding-induced conformational changes in the representative calcium sensors calmodulin (CaM) and troponin C was performed to obtain insights into the underlying molecular basis for their response to the binding of calcium. Distance difference matrices, analysis of interresidue contacts, comparisons of interhelical angles, and inspection of structures using molecular graphics were used to make unbiased comparisons of the various structures. The calcium-induced conformational changes in these proteins are dominated by reorganization of the packing of the four helices within each domain. Comparison of the closed and open conformations confirms that calcium binding causes opening within each of the EF-hands. A secondary analysis of the conformation of the C-terminal domain of CaM (CaM-C) clearly shows that CaM-C occupies a closed conformation in the absence of calcium that is distinct from the semi-open conformation observed in the C-terminal EF-hand domains of myosin light chains. These studies provide insight into the structural basis for these changes and into the differential response to calcium binding of various members of the EF-hand calcium-binding protein family. Factors contributing to the stability of the Ca2+-loaded open conformation are discussed, including a new hypothesis that critical hydrophobic interactions stabilize the open conformation in Ca2+ sensors, but are absent in "non-sensor" proteins that remain closed upon Ca2+ binding. A role for methionine residues in stabilizing the open conformation is also proposed.

NMR studies of tandem WW domains of Nedd4 in complex with a PY motif-containing region of the epithelial sodium channel.

Biochem Cell Biol. 1998; 76: 341-50

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Nedd4 (neuronal precursor cell-expressed developmentally down-regulated 4) is a ubiquitin-protein ligase containing multiple WW domains. We have previously demonstrated the association between the WW domains of Nedd4 and PPxY (PY) motifs of the epithelial sodium channel (ENaC). In this paper, we report the assignment of backbone 1H alpha, 1HN, 15N, 13C', 13C alpha, and aliphatic 13C resonances of a fragment of rat Nedd4 (rNedd4) containing the two C-terminal WW domains, WW(II+III), complexed to a PY motif-containing peptide derived from the beta subunit of rat ENaC, the betaP2 peptide. The secondary structures of these two WW domains, determined from chemical shifts of 13C alpha and 13C beta resonances, are virtually identical to those of the WW domains of the Yes-associated protein YAP65 and the peptidyl-prolyl isomerase Pin1. Triple resonance experiments that detect the 1H alpha chemical shift were necessary to complete the chemical shift assignment, owing to the large number of proline residues in this fragment of rNedd4. A new experiment, which correlates sequential residues via their 15N nuclei and also detects 1H alpha chemical shifts, is introduced and its utility for the chemical shift assignment of sequential proline residues is discussed. Data collected on the WW(II+III)-betaP2 complex indicate that these WW domains have different affinities for the betaP2 peptide.

The calcium-induced structural changes in the skeletal muscle regulatory protein troponin C involve a transition from a closed to an open structure with the concomitant exposure of a large hydrophobic interaction site for target proteins. NMR solution structural studies have served to define this conformational change and elucidate the mechanism of the linkage between calcium binding and the induced structural changes. These structural movements are described in terms of interhelical angles in these largely helical proteins. Oddly, the most recent structure of the cardiac system challenges the central paradigm because the calcium-bound structures are not open. The kinetics, energetics, and dynamics of these proteins have also been investigated using NMR.

New perspectives on S100 proteins: a multi-functional Ca(2+)-, Zn(2+)- and Cu(2+)-binding protein family.

Biometals. 1998; 11: 383-97

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S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcellular localizations and relocations, of post-translational modifications, and of affinities for Ca2+, Zn2+, and Cu2+, consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn2+ and Cu2+, rather than by Ca2+. Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca2+ binding and two lateral alpha-helices III and III, which project outward of each subunit when Ca2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca2+. The selectivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic alpha-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.

Electrostatic coupling to pH-titrating sites as a source of cooperativity in protein-ligand binding.

Protein Sci. 1998; 7: 2012-25

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This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligand-binding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin D9k, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin D9k, the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.

S100 proteins are a group of small dimeric calcium-binding proteins making up a large subclass of the EF-hand family of calcium-binding proteins. Members of this family of proteins have been proposed to act as intracellular calcium modulatory proteins in a fashion analogous to that of the EF-hand sensor proteins troponin-C and calmodulin. Recently, NMR spectroscopy has provided the three-dimensional structures of the S100 family members S100A6 and S100B in both the apo- and calcium-bound forms. These structures have allowed for the identification of a novel calcium-induced conformational change termed the change-in-hand mechanism. Helix III of the C-terminal calcium-binding loop changes its helix-helix interactions (or handness) with the remainder of the molecule primarily owing to the reorientation of the backbone in an effort to coordinate the calcium ion. This reorientation of helix III exposes several residues in the C-terminus and linker regions of S100B resulting in the formation of a hydrophobic patch surrounded be a number of acidic residues. This site is the proposed region for protein-protein recognition.

Calcium-binding proteins in the retina of a calbindin-null mutant mouse.

Cell Tissue Res. 1998; 292: 211-8

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Calcium-binding proteins are abundantly expressed in many neurons of mammalian retinae. Their physiological roles are, however, largely unknown. This is particularly true for calcium-modulating proteins ("calcium buffers") such as calbindin D28k. Here, we have studied retinae of wildtype (+/+) and calbindin-null mutant (-/-) mice by using immunocytochemical methods. Although calbindin immunoreactivity was completely absent in the calbindin (-/-) retinae, those cells that express the protein in wildtype retinae, such as horizontal cells, were still present and appeared normal. This was verified by immunostaining horizontal cells for various neurofilament proteins. In order to assess whether other calcium-binding proteins are upregulated in the mutant mouse and may thus compensate for the loss of calbindin, mouse retinae were also immunolabeled for parvalbumin, calretinin, and a calmodulin-like protein (CALP). In no instance could a change in the expression pattern of these proteins be detected by immunocytochemical methods. Thus, our results show that calbindin is not required for the maintenance of the light-microscopic structure of the differentiated retina and suggest roles for this protein in retinal function.

To investigate the roles of the receptor-dependent actions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in the regulation of vitamin D-dependent calcium-binding proteins (calbindin-D), the messenger RNA (mRNA) levels of calbindin-D9k and -28k were examined in vitamin D receptor (VDR)-ablated mice and control littermates. In VDR-ablated mice, calbindin-D9k mRNA was dramatically reduced in the intestine, kidneys, lungs, and brain; however, calbindin-D28k mRNA was only moderately decreased in the kidney. After 1,25-(OH)2D3 injection, calbindin-D9k mRNA levels and renal and alveolar calbindin-D28k mRNA levels were induced in control animals, but not in the homozygous mice. When the mice were fed a diet high in lactose, calcium, and phosphorus, intestinal calbindin-D9k mRNA levels in the homozygous mice were restored to those in their control littermates. However, this diet failed to normalize extraintestinal calbindin mRNA levels. These findings demonstrate that the receptor-dependent actions of 1,25-(OH)2D3 regulate calbindin-D9k gene expression and that tissue-specific factors modulate the effects of 1,25-(OH)2D3 on calbindin-D28k gene expression. These data also demonstrate that in the absence of a functional VDR, a high local concentration of calcium, phosphorus, and/or lactose in the intestinal lumen can normalize intestinal calbindin-D9k mRNA levels.

Crystal structure of troponin C in complex with troponin I fragment at 2.3-A resolution.

Proc Natl Acad Sci U S A. 1998; 95: 4847-52

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Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca2+-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca2+ bound state) in complex with the N-terminal fragment of TnI (TnI1-47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (Rfree = 32.6%). The central, connecting alpha-helix observed in the structure of uncomplexed TnC (TnCfree) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90 degrees. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI1-47 alpha-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI1-47 alpha-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca2+ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI1-47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique alpha-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca2+ bound conformation.

Several EF-hand recoverin mutants were obtained and their abilities to bind to photoreceptor membranes and to inhibit rhodopsin kinase were determined. The mutants with the 'spoiled' 2nd, 3rd or (2nd+3rd) EF-hand structures did not act upon the kinase activity in the microM range of Ca2+ concentrations. Mutations of the 4th EF hand, which 'repaired' its Ca2+-binding activity, resulted in recoverin with three 'working' Ca2+-binding sites. The latter mutant inhibited rhodopsin kinase even more effectively than the wild-type recoverin, containing two working Ca2+-binding structures.

The identification and differential expression of calcium-binding proteins associated with ocular melanoma.

Biochim Biophys Acta. 1998; 1448: 290-7

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Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.

We have identified and characterized a cDNA encoding a novel FK506-binding protein (FKBP), named FKBP23, from mouse heart by the signal sequence trap method. The deduced amino acid sequence has significant homology to other FKBP family members around the peptidylprolyl cis-trans-isomerase motifs. FKBP23 also has two Ca2+-binding (EF-hand) motifs, and purified FKBP23 protein was shown to have Ca2+-binding ability. This is the first report of a Ca2+-binding FKBP. FKBP23 is a glycoprotein retained in the endoplasmic reticulum by its carboxyl-terminal tetrapeptide His-Asp-Glu-Leu, as demonstrated by immunostaining, retention, and deglycosylation assays. FKBP23 mRNA is expressed most strongly in heart, lung, and testis, beginning at day 8.5 of embryonic development. The FKBP23 gene was mapped to mouse chromosome 2.

We have generated a series of chicken skeletal muscle troponin C mutants to study the conformation of the regulatory domain in the N-terminal half of the molecule. These mutants each contained a single Trp at position 22 (helix A), 52 (linker of helices B and C), or 90 (central helix). Some of these mutants also contained additional mutations to introduce a single Cys at a desired position. The mutants were characterized by molecular graphics and CD and found to have a minimum of structural perturbations when compared with the native structure. They also retained the ability to regulate myofibrillar ATPase activity. The fluorescence of Trp22 was sensitive to Ca2+ binding only to the regulatory sites, whereas Trp52 and Trp90 responded to Ca2+ binding to both the regulatory and the Ca2+/Mg2+ sites. The tryptophan quantum yield (Q) of all Trp22-containing mutants was very high (0.33) in the absence of bound Ca2+, compared to that of L-tryptophan in aqueous solution (0.14). Q decreased 25% upon binding of Ca2+ to the regulatory sites. The quantum yields of Trp52 and Trp90 in apo mutants were close to 0.14. In the presence of bound Ca2+ at the regulatory sites, the quantum yield of Trp52 decreased 16%, whereas that of Trp90 increased 25%. Results from acrylamide quenching of the fluorescence of the three Trp residues indicated that Trp22 was the least exposed and Trp52 was the most exposed, consistent with other spectral data that Trp22 was in a relatively nonpolar environment and Trp52 was in a highly polar environment. The ability of Trp52 and Trp90 to sense Ca2+ binding to sites located at both domains suggests inter-domain communication in the protein. These single Trp TnC mutants provide specific signals for probing Ca2+-induced conformational changes in the regulatory domain.

The effects of hydrophobic core mutations on the stability and structure of the four-helix calcium-binding protein, calbindin D9k, have been investigated. Eleven mutations involving eight residues distributed within the hydrophobic core of calbindin D9k were examined. Stabilities were measured by denaturant and thermal induced unfolding monitored by circular dichroism spectroscopy. The mutations were found to exert large effects on the stability with midpoints in the urea induced unfolding varying from 1.8 M for Leu23 --> Gly up to 6.6 M for Val70 --> Leu and free energies of unfolding in the absence of denaturant ranging from 6.6 to 27.4 kJ/mol for the Phe66 --> Ala mutant and the wild-type, respectively. A significant correlation was found between the difference in free energy of unfolding (Delta Delta GNU) and the change in the surface area of the side chain caused by the mutation, in agreement with other studies. Notably, both increases and decreases in side-chain surface area caused quantitatively equivalent effects on the stability. In other words, a correlation between the absolute value of the change in the surface of the side chain and Delta DeltaGNU was observed with a value of approximately 0.14 kJ M-1 A-2. The generality of this observation is discussed. Significant effects on the cooperativity of the unfolding reaction were also observed. However, a correlation between the cooperativity and Delta Delta GNU, which has been reported in other systems as an indication of effects of mutations on the unfolded state, was not observed for calbindin D9k. Despite the large effects on Delta Delta GNU and cooperativity, the structures of the mutants in the native form remained intact as indicated by circular dichroism, NMR, and fluorescence measurements. The structural response to calcium-binding was also conserved. The following paper in this issue [Kragelund, B. B., et al. (1998) Biochemistry 37, 8926-8937] examines the effects of these mutations on the calcium binding properties of calbindin D9k.

Calcium-binding proteins contain a variable number of motifs, termed EF-hands, which consist of two perpendicularly placed alpha-helics and an inter-helical loop forming a single calcium-binding site. Due to their ability to bind and transport calcium as well as to interact with a variety of ligands in a calcium-dependent manner, they fulfill important biological functions in eukaryotic cells. After parvalbumin, a three EF-hand fish allergen, calcium-binding allergens were discovered in pollens of trees. grasses and weeds and, recently, as autoallergens in man. Although only a small percentage of atopic individuals displays IgE reactivity to calcium-binding allergens, these allergens may be important because of their ability to cross-sensitize allergic individuals. Confrontation and stability++ as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. It is thus likely that hypoallergenic derivatives of calcium-binding allergens can be engineered by recombinant DNA technology for immunotherapy++ of sensitized patients.

GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.

Calcium- and pH-linked oligomerization of sorcin causing translocation from cytosol to membranes.

FEBS Lett. 1997; 409: 1-6

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Sorcin, a cytosolic calcium-binding protein containing a pair of EF-hand motifs, undergoes a Ca2(+)-dependent translocation to the cell membrane. The underlying conformational change is similar at pH 6.0 and 7.5 and consists in an increase in overall hydrophobicity that involves the aromatic residues and in particular the two tryptophan residues which become less exposed to solvent. The concomitant association from dimers to tetramers indicates that the tryptophan residues, which are located between the EF-hand sites, become buried at the dimer-dimer interface. Ca2(+)-bound sorcin displays a striking difference in solubility as a function of pH that has been ascribed to the formation of calcium-stabilized aggregates.

A calcium responsive element that regulates expression of two calcium binding proteins in Purkinje cells.

Proc Natl Acad Sci U S A. 1997; 94: 8842-7

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Calbindin D28 encodes a calcium binding protein that is expressed in the cerebellum exclusively in Purkinje cells. We have used biolistic transfection of organotypic slices of P12 cerebellum to identify a 40-bp element from the calbindin promoter that is necessary and sufficient for Purkinje cell specific expression in this transient in situ assay. This element (PCE1) is also present in the calmodulin II promoter, which regulates expression of a second Purkinje cell Ca2+ binding protein. Expression of high levels of exogenous calbindin or calretinin decreased transcription mediated by PCE1 in Purkinje cells 2.5- to 3-fold, whereas the presence of 1 microM ionomycin in the extracellular medium increased expression. These results demonstrate that PCE1 is a component of a cell-specific and Ca2+-sensitive transcriptional regulatory mechanism that may play a key role in setting the Ca2+ buffering capacity of Purkinje cells.

S100 beta is a member of a group of low-molecular weight acidic calcium binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and II). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy using Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characterize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta shows two distinct Eu3+ binding environments from both the excitation spectrum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd of 660 +/- 20 nM, whereas the dissociation constant for the pseudo-EF hand site I is significantly weaker. Lifetimes in H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water molecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln3+ binding domains I and II in calmodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta show that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is significantly different than that in the absence of Zn2+. Tyrosine-17-sensitized Tb3+ luminescence experiments indicate that the Tb3+ occupying the proximal weaker binding site I is sensitized, whereas Tb3+ in site II is not. The distance between sites I and II (15.0 +/- 0.4 A) in S100 beta was determined from Forster-type energy transfer in D2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Zn2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 A. Location of histidine-15 close to pseudo-EF site I suggests that Zn2+ binding likely changes the conformation of this site, causing a reduction of the intersite distance by approximately 2 A.

Recoverin is an EF-hand calcium-binding protein reportedly involved in the transduction of light by vertebrate photoreceptor cells. It also is an autoantigen in a cancer-associated degenerative disease of the retina. Measurements by circular dichroism presented here demonstrate that the binding of calcium to recoverin causes large structural changes. increasing the alpha-helical content of the protein and decreasing its beta-turn, beta-sheet and 'other' structures. The maximum helical content (67%) was observed at 100 microM free calcium and, unlike calmodulin, decreased as the calcium concentration was modulated in either direction from this value. Fluorescence measurements indicated that recoverin may aggregate or undergo structural changes independent of calcium binding as the calcium concentration is increased above 100 microM. EGTA also appeared to affect the structure of recoverin independent of its chelation of calcium. While calcium-induced conformational changes have been proposed to alter the membrane binding of recoverin through association of its myristoylated amino terminus, in the experiments presented here the partitioning of recoverin between the cytoplasmic and membrane compartments of the rod photoreceptor outer segment was unaffected by the concentration of calcium, therefore it appears unlikely that a calcium-myristoyl switch acts alone to anchor recoverin directly to the membrane. These experiments were conducted with native recoverin which is heterogeneously acylated, but mass spectrometry confirmed that simple chromatographic methods could be devised to isolate the different forms of recoverin for further studies.

Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments.

Protein Sci. 1997; 6: 2385-96

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Calbindin D28k is an intracellular Ca(2+)-binding protein containing six subdomains of EF-hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF-hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion-exchange chromatography. It was anticipated that if the peptides associate to form native-like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k, suggesting that the mixed EF-hand fragments associate to form a native-like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF-hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF-hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.

Is the structure of the N-domain of phosphoglycerate kinase affected by isolation from the intact molecule?

Biochemistry. 1997; 36: 333-40

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The structural integrity of the isolated N-domain (residues 1-174) of Bacillus stearothermophilus 3-phosphoglycerate kinase (PGK) has been investigated using heteronuclear NMR spectroscopy. Analysis of 13C chemical shifts, amide protection, and comparison of observed and expected sequential NOE intensities calculated from the crystal structure of the domain in the intact protein indicate that the secondary structure of the isolated domain is unchanged from that found in the intact molecule. Markedly shifted 1H resonances, amide protection, and long-range NOEs indicate that the tertiary structure is similarly unaffected. These results are confirmed by an excellent agreement (standard deviation 0.28 ppm) between observed H alpha chemical shifts and those calculated from the high-resolution (1.6 A) crystal structure of intact PGK [Davies et al. (1994) Acta Crystallogr. D50, 202-209]. The only region perturbed by loss of interactions with the C-domain is a small portion of the substrate-binding site (residues 148-152) whose amide protons are poorly protected from solvent. These results provide a structural basis for the analysis of the folding of the domains of PGK as isolated units and within the intact molecule [Parker et al. (1996) Biochemistry (in press)] and contrast with the notion that the native tertiary fold of the N-domain of PGK requires the whole polypeptide chain, including the entire C-domain [Mas et al. (1995) Biochemistry 34, 7931-7940]. Assignments of backbone 13C, 15N, HN, and H alpha resonances are provided.

High resolution solution structure of a DNA duplex alkylated by the antitumor agent duocarmycin SA.

J Mol Biol. 1997; 272: 237-52

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The three-dimensional solution structure of duocarmycin SA in complex with d-(G1ACTAATTGAC11).d-(G12TCATTAGTC22) has been determined by restrained molecular dynamics and relaxation matrix calculations using experimental NOE distance and torsion angle constraints derived from 1H NMR spectroscopy. The final input data consisted of a total of 858 distance and 189 dihedral angle constraints, an average of 46 constraints per residue. In the ensemble of 20 final structures, there were no distance constraint violations >0.06 A or torsion angle violations >0.8 degrees. The average pairwise root mean square deviation (RMSD) over all 20 structures for the binding site region is 0.57 A (average RMSD from the mean: 0.39 A). Although the DNA is very B-like, the sugar-phosphate backbone torsion angles beta, epsilon, and zeta are distorted from standard values in the binding site region. The structure reveals site-specific bonding of duocarmycin SA at the N3 position of adenine 19 in the AT-rich minor groove of the duplex and binding stabilization via hydrophobic interactions. Comparisons have been made to the structure of a closely related complex of duocarmycin A bound to an AT-rich DNA duplex. These results provide insights into critical aspects of the alkylation site selectivity and source of catalysis of the DNA alkylating agents, and the unusual stability of the resulting adducts.

While calcium binding to troponin C (TnC) triggers the contraction of both skeletal and cardiac muscle, there is clear evidence that different mechanisms may be involved. For example, activation of heart myofilaments occurs with binding to a single regulatory site on TnC, whereas activation of fast skeletal myofilaments occurs with binding to two regulatory sites. The physiological difference between activation of cardiac and skeletal myofilaments is not understood at the molecular level due to a lack of structural details for the response of cardiac TnC to calcium. We determined the solution structures of the apo and calcium-saturated regulatory domain of human cardiac TnC by using multinuclear, multidimensional nuclear magnetic resonance spectroscopy. The structure of apo human cardiac TnC is very similar to that of apo turkey skeletal TnC even though there are critical amino acid substitutions in site I. In contrast to the case with the skeletal protein, the calcium-induced conformational transition in the cardiac regulatory domain does not involve an "opening" of the regulatory domain, and the concomitant exposure of a substantial hydrophobic surface area. This result has important implications with regard to potential unique aspects of the interaction of cardiac TnC with cardiac troponin I and of modification of cardiac myofilament regulation by calcium-sensitizer drugs.

Structural interactions responsible for the assembly of the troponin complex on the muscle thin filament.

Cell Struct Funct. 1997; 22: 219-23

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Skeletal muscle contraction is regulated by a complex of five polypeptides which are stably associated with the actin filament. This complex consists of two proteins: troponin with three subunits (TnC; TnI and TnT) and tropomyosin (a dimer of two chains). Using deletion mutants of TnC, TnI and TnT we determined that each of these polypeptides can be divided into at least two domains. One domain is responsible for the regulatory properties of the protein. Its interaction with the other components of the system change upon calcium binding to TnC. A second domain present in each of these proteins is responsible for the stable association of the complex to the actin filament. The interactions among this second set of domains is not influenced by calcium binding to TnC. The structural interactions are: 1) interactions between the C-domain of TnC with the N-domain of TnI; 2) interactions of the N-domain of TnI with the C-terminal domain of TnT and 3) interactions between the N-domain of TnT (T1) and actin/tropomyosin.

Many eukaryotic cellular and viral proteins have a covalently attached myristoyl group at the amino terminus. One such protein is recoverin, a calcium sensor in retinal rod cells, which controls the lifetime of photoexcited rhodopsin by inhibiting rhodopsin kinase. Recoverin has a relative molecular mass of 23,000 (M[r] 23K), and contains an amino-terminal myristoyl group (or related acyl group) and four EF hands. The binding of two Ca2+ ions to recoverin leads to its translocation from the cytosol to the disc membrane. In the Ca2+-free state, the myristoyl group is sequestered in a deep hydrophobic box, where it is clamped by multiple residues contributed by three of the EF hands. We have used nuclear magnetic resonance to show that Ca2+ induces the unclamping and extrusion of the myristoyl group, enabling it to interact with a lipid bilayer membrane. The transition is also accompanied by a 45-degree rotation of the amino-terminal domain relative to the carboxy-terminal domain, and many hydrophobic residues are exposed. The conservation of the myristoyl binding site and two swivels in recoverin homologues from yeast to humans indicates that calcium-myristoyl switches are ancient devices for controlling calcium-sensitive processes.

The actomyosin ATPase inhibitory protein troponin I (TnI) plays a central regulatory role in skeletal and cardiac muscle contraction and relaxation through its calcium-dependent interactions with troponin C (TnC) and actin. Previously we have demonstrated the utility of F29W and F105W mutants of TnC for measurement of binding affinities of inhibitory peptide TnI(96-116) to its regulatory N and structural C domains, both in isolation and in the intact TnC molecule [Pearlstone, J. R. & Smillie, L. B. (1995) Biochemistry 34, 6932-6940]. This approach is now extended to fragment TnI(96-148). Curve-fitting analyses of fluorescence changes induced in the intact TnC mutants and the isolated N and C domains by increasing [TnI(96-148)] have permitted the assignments of K(D) values (designated K(D,N) and K(D,C)) to the interaction of TnI(96-148) with the N and C domains, respectively, of intact TnC. Taken together with the previous data for TnI(96-116) binding, it can be concluded that, within TnI(96-148), residues 96-116 are primarily responsible for binding to C domain of intact TnC and residues 117-148 to its N domain. Inspection of the available mammalian and avian skeletal muscle TnI amino acid sequences reveals a previously unrecognized conserved motif repeated 3-fold, once in the inhibitory peptide region (approximately residues 101-114; designated alpha) and twice more in the region of residues approximately 121-132 (beta) and approximately 135-146 (gamma). The number and distribution of these motifs have important structural implications for the TnI x C complex. In the beta motif of cardiac TnI, as compared with skeletal, several changes in charged amino acids are suggested as candidates responsible for the greater sensitivity of cardiac Ca2+-regulated actomyosin to acidic pH as in ischemia.

Chick calretinin has been previously expressed in Escherichia coli and purified to homogeneity [Cheung, W-T., Richards, D.E., and Rogers, J.H. (1993) Eur. J. Biochem. 215, 401-410]. In the present study we have developed an improved purification procedure, involving a heat precipitation step followed by DEAE-cellulose chromatography with calcium-dependent elution. Native calretinin was purified from chick brainstem using the same method as for the recombinant protein but with an added affinity chromatography step. Typically 30 g of brainstem yielded 350 micrograms of protein. Several differences between the two forms imply that the native protein is acetylated at the N-terminus but otherwise unmodified. The calcium binding activities of both forms of calretinin were measured by equilibrium dialysis with 45Ca in Ca2+/EGTA buffers. The recombinant form bound 4.9 +/- 0.12 calcium ions with Kd = 0.38 +/- 0.02 microM and the native form was not significantly different. Recombinant calretinin was used to study its interaction with other cations present in cells and it was found that calcium binding was affected by Mg2+. Calretinin appears to bind 4.69 +/- 0.13 magnesium ions with Kd = 4.5 mM. Mg2+ increased the apparent dissociation constant for Ca2+. The shift is consistent with competitive binding of Ca2+ and Mg2+ to the same five sites, but Mg2+ binding is too weak to interfere significantly with Ca2+ binding under physiological conditions.

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

Protein Sci. 1997; 6: 1139-47

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The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.

Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.

Reticulocalbin (RCN) is a member of the EF-hand Ca(2+)-binding protein family and is a luminal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Although RCN has six repeats of a domain containing an EF-hand motif, the varying degrees of divergence of the amino acid sequences of these domains from the EF-hand consensus sequences suggested that some domains might have lost their Ca(2+)-binding capability and adopted new functions. To identify the domains involved in Ca(2+)-binding, discrete domains of RCN were expressed in Escherichia coli, using the glutathione S-transferase fusion protein system. 45Ca2+ blot analysis of the resultant fusion proteins revealed that the first, fourth, fifth, and sixth domains bind Ca2+, however, the second and third ones do not. The fusion proteins containing all six domains, and the first and second domains, respectively, showed Ca(2+)-dependent increases in their electrophoretic mobilities, suggesting that Ca2+ induces a conformational change in reticulocalbin.

Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution.

J Mol Biol. 1997; 273: 238-55

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We have solved and refined the crystal and molecular structures of the calcium-saturated N-terminal domain of troponin C (TnC) to 1.75 A resolution. This has allowed for the first detailed analysis of the calcium binding sites of this molecular switch in the calcium-loaded state. The results provide support for the proposed binding order and qualitatively, for the affinity of calcium in the two regulatory calcium binding sites. Based on a comparison with the high-resolution apo-form of TnC we propose a possible mechanism for the calcium-mediated exposure of a large hydrophobic surface that is central to the initiation of muscle contraction within the cell.

Calcium is involved in most cellular processes through calcium-binding proteins, belonging to the EF-hand superfamily. Recently new members of this family were found in the retina of vertebrates. Functions of these proteins and their relations to some neurological diseases are reviewed. Other EF-hand proteins found in the retina are also described.

Current methods of determining the rotational diffusion tensors of proteins in solution by NMR spectroscopy exclusively utilize relaxation rate constants for backbone amide 15N spins. However, the distributions of orientations of N-H bond vectors are not isotropic in many proteins, and correlations between bond vector orientations reduce the accuracy and precision of rotational diffusion tensors extracted from 15N spin relaxation data. The inclusion of both 13C alpha and 15N spin relaxation rate constants increases the robustness of the diffusion tensor analysis because the orientations of the C alpha-H alpha bond vectors differ from the orientations of the N-H bond vectors. Theoretical and experimental results for calbindin D9k, granulocyte colony stimulating factor, and ubiquitin, three proteins with different distributions of N-H and C alpha-H alpha bond vectors, are used to illustrate the advantages of the simultaneous utilization of 13C alpha and 15N relaxation data.

The three-dimensional solution structures of proteins determined with NMR-derived constraints are almost always calculated in vacuo. The solution structure of (Ca2+)2-calbindin D9k has been redetermined by new restrained molecular dynamics (MD) calculations that include Ca2+ ions and explicit solvent molecules. Four parallel sets of MD refinements were run to provide accurate comparisons of structures produced in vacuo, in vacuo with Ca2+ ions, and with two different protocols in a solvent bath with Ca2+ ions. The structural ensembles were analyzed in terms of structural definition, molecular energies, packing density, solvent-accessible surface, hydrogen bonds, and the coordination of calcium ions in the two binding loops. Refinement including Ca2+ ions and explicit solvent results in significant improvements in the precision and accuracy of the structure, particularly in the binding loops. These results are consistent with results previously obtained in free MD simulations of proteins in solution and show that the rMD refined NMR-derived solution structures of proteins, especially metalloproteins, can be significantly improved by these strategies.

Phenol hydroxylase from Pseudomonas sp. CF600 is a member of a family of binuclear iron-center-containing multicomponent oxygenases, which catalyzes the conversion of phenol and some of its methyl-substituted derivatives to catechol. In addition to a reductase component which transfers electrons from NADH, optimal turnover of the hydroxylase requires P2, a protein containing 90 amino acids which is readily resolved from the other components. The three-dimensional solution structure of P2 has been solved by 3D heteronuclear NMR spectroscopy. On the basis of 1206 experimental constraints, including 1060 distance constraints obtained from NOEs, 70 phi dihedral angle constraints, 42 psi dihedral angle constraints, and 34 hydrogen bond constraints, a total of 12 converged structures were obtained. The atomic root mean square deviation for the 12 converged structure with respect to the mean coordinates is 2.48 A for the backbone atoms and 3.85 A for all the heavy atoms. This relatively large uncertainty can be ascribed to conformational flexibility and exchange. The molecular structure of P2 is composed of three helices, six antiparallel beta-strands, one beta-hairpin, and some less ordered regions. This is the first structure among the known multicomponent oxygenases. On the basis of the three-dimensional structure of P2, sequence comparisons with similar proteins from other multicomponent oxygenases suggested that all of these proteins may have a conserved structure in the core regions.

The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.

A sequential assignment protocol for proteins was developed using heteronuclear 3D NMR. The protocol consists of an amino acid type recognition algorithm and a primary sequence mapping algorithm. The former measures the similarity between each detected spin pattern and 20 standard amino acid coupling patterns. Both chemical shift and topologically likeness are considered. The mapping algorithm uses the amino acid type information to direct detected polypeptides to proper position onto protein primary sequence. The assignment protocol can be applied to spin systems generated by many different approaches. We designed a few computer programs to derive a protein's backbone and side chain spin systems using heteronuclear 3D NMR. The results was then input to the sequential assignment protocol. All of the algorithms were tested on NMR data of a 90-residue N-domain of chicken skeletal troponin-C.

Structures of four Ca2+-bound troponin C at 2.0 A resolution: further insights into the Ca2+-switch in the calmodulin superfamily.

Structure. 1997; 5: 1695-711

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BACKGROUND: In contrast to Ca2+4-bound calmodulin (CaM), which has evolved to bind to many target sequences and thus regulate the function of a variety of enzymes, troponin C (TnC) is a bistable switch which controls contraction in striated muscles. The specific target of TnC is troponin I (TnI), the inhibitory subunit of the troponin complex on the thin filaments of muscle. To date, only the crystal structure of Ca2+2-bound TnC (i.e. in the 'off' state) had been determined, which together with the structure of Ca2+4-bound CaM formed the basis for the so-called 'HMJ' model of the conformational changes in TnC upon Ca2+ binding. NMR spectroscopic studies of Ca2+4-bound TnC (i.e. in the 'on' state) have recently been carried out, but the detailed conformational changes that take place upon switching from the off to the on state have not yet been described. RESULTS: We have determined the crystal structures of two forms of expressed rabbit Ca2+4-bound TnC to 2.0 A resolution. The structures show that the conformation of the N-terminal lobe (N lobe) is similar to that predicted by the HMJ model. Our results also reveal, in detail, the residues involved in binding of Ca2+ in the regulatory N lobe of the molecule. We show that the central helix, which links the N and C lobes of TnC, is better stabilized in the Ca2+2-bound than in the Ca2+4-bound state of the molecule. Comparison of the crystal structures of the off and on states of TnC reveals the specific linkages in the molecule that change in the transition from off to on state upon Ca2+-binding. Small sequence differences are also shown to account for large functional differences between CaM and TnC. CONCLUSIONS: The two lobes of TnC are designed to respond to Ca2+-binding quite differently, although the structures with bound Ca2+ are very similar. A small number of differences in the sequences of these two lobes accounts for the fact that the C lobe is stabilized only in the open (Ca2+-bound) state, whereas the N lobe can switch between two stable states. This difference accounts for the Ca2+-dependent and Ca2+-independent interactions of the N and C lobe. The C lobe of TnC is always linked to TnI, whereas the N lobe can maintain its regulatory role - binding strongly to TnI at critical levels of Ca2+ - and in contrast, forming a stable closed conformation in the absence of Ca2+.

Cloning and characterization of a calcium-sensing receptor from the hypercalcemic New Zealand white rabbit reveals unaltered responsiveness to extracellular calcium.

J Bone Miner Res. 1997; 12: 568-79

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The extracellular Ca2+ (Ca(0)2+)-sensing receptor (CaR) recently cloned from mammalian parathyroid, kidney, brain, and thyroid plays a central role in maintaining near constancy of Ca(0)2+. We previously showed that the hypercalcemia normally present in New Zealand white rabbits is associated with an elevated set point for Ca(02+)-regulated PTH release (the level of Ca(0)2+ half-maximally inhibiting hormonal secretion). This observation suggested an alteration in the Ca(02+)-sensing mechanism in the rabbit parathyroid, a possibility we have now pursued by isolating and characterizing the rabbit homolog of the CaR. The cloned rabbit kidney CaR (RabCaR) shares a high degree of overall homology (> 90% amino acid identity) with the bovine, human, and rat CaRs, although it differs slightly in several regions of the extracellular domain potentially involved in binding ligands. By Northern analysis and/or immunohistochemistry, a similar or identical receptor is also expressed in parathyroid, thyroid C cells, small and large intestine, and in the thick ascending limb and collecting ducts of the kidney. When expressed transiently in HEK293 cells and assayed functionally through CaR agonist-evoked increases in Ca(i)2+, the rabbit CaR shows apparent affinities for Ca(0)2+, Mg(0)2+, and Gd(0)3+ that are indistinguishable from those observed in studies carried out concomitantly using the human CaR. Therefore, at least as assessed by its ability to increase Ca(i)2+ when expressed in HEK293 cells, the intrinsic functional properties of the rabbit CaR cannot explain the hypercalcemia observed in vivo in the New Zealand white rabbit.

Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.

The three-dimensional solution structure of plastocyanin from Anabaena variabilis (A.v.PCu) has been determined by nuclear magnetic resonance spectroscopy. Sixty structures were calculated by distance geometry from 1141 distance restraints and 46 dihedral angle restraints. The distance geometry structures were optimized by simulated annealing and restrained energy minimization. The average rms deviation from the mean structure for the 20 structures with the lowest total energy is 1.25 A for the backbone atoms and 1.75 A for all heavy atoms. Overall, the global tertiary fold of A.v.PCu resembles those of other plastocyanins which have been structurally characterized by X-ray diffraction and NMR methods. This holds even though A.v.PCu is longer than any other known plastocyanins, contains far less invariant amino acid residues, and has an overall charge that differs considerably from those of other plastocyanins (+1 vs -9 +/- 1 at pH > or = 7). The most striking feature of the A.v. PCu structure is the absence of the beta-turn, formed at the remote site by residues (58)-(61) in most higher plant plastocyanins. The displacement caused by the absence of this turn is compensated for by an extension of the small helix [from Ala53(51) to Ser60(58) in A.v.PCu] found in other plastocyanins. Moreover, the extra residues of A.v.PCu from Pro77 to Asp79 form an appended loop. These two features allow A.v.PCu to retain almost the same global fold as observed in other plastocyanins. From a comparison with the structures of other plastocyanins it is concluded that the lack of negatively charged residues at the remote site, rather than the specific structure of A.v.PCu, is the main reason for the failure of the remote site of this plastocyanin to function as a significant electron transfer site.

Measurement and modelling of sequence-specific pKa values of lysine residues in calbindin D9k.

J Mol Biol. 1996; 259: 828-39

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A pH titration study of calbindin D9k was performed using heteronuclear 1H-13C two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to the titrating group. The site-specific pKa values of these lysine residues, ranging from 10.1 to 12.1, were obtained from the analysis of pH-dependent chemical shifts of 13C and 1H resonances. Ionization constants for both the Ca(2+)-free (apo) and Ca(2+)-loaded forms of the protein were determined. The proton uptake by lysine residues in the apo form was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the pKa values of all lysine residues. The largest reduction of one pK unit was observed for Lys55, which is also the closest to the calcium binding sites. A threefold increase in protein concentration, from 0.5 to 1.5 mM, reduced the pKa values by 0.1 to 0.4 pK unit in agreement with the screening concept of ionic interactions. All the observed pKa shifts were site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensional structure of calbindin D9k and a dielectric continuum model for the electrostatic interactions.

[Study of calcium-binding properties of the Mts-1 protein and its recombinant analog using the fluorescent probe Fura-2]

Biokhimiia. 1996; 61: 919-26

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A procedure based on the ability of Mts-1 to competitively inhibit the formation of Fura-2 complexes with Ca(2+)-ions is described. It has been shown that Mts-I reversibly inhibits the complex formation between Fura-2 and calcium. The efficiency of natural Mts-I isolated from cells of the CSML-100 line and of its recombinant analog on the formation of the Fura-2+Ca complex is different. The inhibition constant, Ki, for the native protein is equal to 2,4 microM, that for the recombinant form is 8.5 microM. The data obtained are suggestive of posttranslational modification of Mts-I in cells under in vivo conditions.

The applications of NMR spectroscopy to muscle proteins are reviewed with particular emphasis on the special problems encountered in muscle systems. Recent applications to the determination of the solution structures of calcium-binding regulatory proteins are highlighted.

Calcium-dependent binding of S100C to the N-terminal domain of annexin I.

J Biol Chem. 1996; 271: 719-25

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The annexin family of proteins is characterized by a conserved core domain that binds to phospholipids in a Ca(2+)-dependent manner. Each annexin also has a structurally distinct N-terminal domain that may impart functional specificity. To search for cellular proteins that interact with the N-terminal domain of annexin I, we constructed a fusion protein consisting of glutathione S-transferase fused to amino acids 2-47 of human annexin I (GST-AINT; AINT = annexin I N-terminal). Extracts from metabolically labeled A431 cells contained a single protein (M(r) approximately 10,000) that bound to GST-AINT in a Ca(2+)-dependent manner. A synthetic peptide corresponding to amino acids 2-18 of annexin I inhibited the binding of the 10-kDa protein to GST-AINT with half-maximal inhibition occurring at approximately 15 microM peptide. In cellular extracts, endogenous annexin I and the 10-kDa protein associated in a reversible Ca(2+)-dependent manner. Experiments with other annexins and with N-terminal truncated forms of annexin I indicated that the 10-kDa protein bound specifically to a site within the first 12 amino acids of annexin I. The 10-kDa protein was purified from human placenta by hydrophobic and affinity chromatography. Amino acid sequence analysis indicated that the 10-kDa protein is the human homologue of S100C, a recently identified member of the S100 subfamily of EF-hand Ca(2+)-binding proteins.

Interaction of a troponin I inhibitory peptide with both domains of troponin C.

Biochim Biophys Acta. 1996; 1294: 25-30

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Skeletal muscle contraction is regulated by Ca2+ binding to troponin (Tn), a complex of three proteins attached to the actin-tropomyosin filaments. We have been investigating key interactions of the Ca(2+)-binding protein TnC and the inhibitory protein TnI. Previously, we used 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI, and found that the N-terminal, regulatory domain of TnC formed cross-links to the inhibitory region of TnI (Leszyk, J., Grabarek, Z., Gergely, J. and Collins, J.H. (1990) Biochemistry 29, 299-304). In the present study we have used EDC to form cross-links between TnC and a synthetic peptide, based on residues 104-115 of TnI, which mimics intact TnI in its ability to inhibit actomyosin ATPase activity. Prior to cross-linking, we acetylated the epsilon-amino groups of the nine lysine residues of TnC in order to prevent intramolecular cross-linking. Cross-linked TnC-peptide products were cleaved with CNBr and several proteinases. The resulting cross-linked peptides were purified by HPLC and characterized by amino-acid sequence analysis. Our results indicate that the TnI peptide interacted most strongly with two sites in TnC: Glu-60 and/or Glu-61 in the N-terminal domain, and acidic residue(s) in segment 84-94 of the linker region which connects the N- and C-terminal domains of TnC.

Phosphorylation of MRP14, an S100 protein expressed during monocytic differentiation, modulates Ca(2+)-dependent translocation from cytoplasm to membranes and cytoskeleton.

J Immunol. 1996; 156: 1247-54

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MRP8 and MRP14 are two Ca(2+)-binding proteins expressed in myelomonocytic cells. Complexes of MRP8 and MRP14 colocalize with membranes and intermediate filaments in a Ca(2+)-dependent manner. MRP14, unlike MRP8, exists in two isoforms, the smaller of which (MRP14') has been shown to lack the first four amino acids; both MRP14 and MRP14' are also present as phosphorylated forms. As shown in the present work by metabolic labeling of monocytes with [35S]methionine, MRP14 and MRP14' are translated simultaneously. By PCR analysis we found no evidence for the presence of different mRNA species. Since MRP14 is encoded by a single copy gene, our data indicate that MRP14' formation is due to alternative translation of a single mRNA species. Two-dimensional electrophoresis of [32P]orthophosphate-labeled monocyte proteins followed by Western blotting and autoradiography revealed that the two phosphorylated MRP14 isoforms incorporated the bulk of the radioactivity found in monocytic proteins. Using differential centrifugation we demonstrated the presence of distinct isoform patterns in different subcellular locations. Further, in response to elevated Ca2+ concentrations we observed a preferential translocation of phosphorylated MRP14 isoforms from the cytosol toward membranes and the cytoskeleton. This might be caused by altered calcium binding, and indeed, using isoelectric focusing and 45Ca2+ overlay the MRP14 band containing phosphorylated MRP14 revealed increased Ca2+ binding compared with bands containing other MRP14 isoforms. This represents the first evidence for functional differences in phosphorylated MRP14 isoforms compared with nonphosphorylated MRP14 isoforms. These functional differences suggest that MRP14 represents the regulatory subunit of MRP8/MRP14 complexes.

Relationship between stability and function for isolated domains of troponin C.

Biochemistry. 1996; 35: 14012-26

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Results of spectroscopic thermal and chemical denaturation studies and calcium binding studies are presented for a series of five recombinant chicken troponin C fragments. They were designed to assess the effects of domain isolation, N-helix, and D/E linker helix on stability and calcium affinity. Four of the fragments include the N-terminal regulatory domain and one the C-terminal domain. For the regulatory domain, deletion of the N-helix or the D/E linker decreases the stability of the apo form as measured by delta GN-->U,25. Separation of the domains also decreases the stability. Differences in values of delta GN-->U,25 derived from urea and guanidine hydrochloride studies allowed an estimation of the electrostatic component of the free energy of unfolding. Our measurements provide the first quantitative estimate of the stability for the apo-C-domain (delta GN-->U,25 = -1.8 kcal/mol) which was obtained using the interaction free energy formalism of Schellman. There is an inverse correlation between calcium affinity, binding cooperativity, and stability for all of these homologously structured fragments. The calcium affinity and cooperativity are highest for the unstructured C-domain and lowest for the N-domain which has the highest stability. In view of the direct effects on the folding stability of the apo-N-domain, the N-helix and the bilobed domain organization of TnC are necessarily involved in the fine-tuning of the affinity and cooperativity of calcium binding. Though not directly involved in calcium coordination, these structural features are important for signal transmission by troponin C in the troponin complex.

The S100 Ca(2+)-binding proteins recently became of major interest because of their differential expression in neoplastic tissues, their involvement in metastatic processes, and the clustered organization of at least 10 S100 genes on human chromosome 1q21, a region frequently rearranged in several tumors. As a first attempt towards a specific and differentiated immunohistochemical classification of human tumors, we produced, purified and characterized a number of human recombinant S100 proteins and raised specific polyclonal antibodies. Their distinct cellular and intracellular localization was examined by immunohistochemical methods in normal and cancerogenic human tissues and cell lines. S100A1 and S100A2 can be detected in a few normal tissues only, whereas S100A4, S100A6, and S100B are expressed at higher levels in cancer tissues. In the future, these S100 antibodies will potentially be of great value in cancer diagnosis and therapy.

Calbindin D28k is an intracellular Ca2+-binding protein noted for its abundance and specific distribution in mammalian brain and sensory neurons. This protein contains six putative Ca2+-binding sites, referred to as EF-hands. Due to the presence of the large number of putative sites, previous studies have been unsuccessful in definitively establishing the stoichiometry of Ca2+ binding. We describe a synthetic approach to identify the number of Ca2+-binding sites in which 6 33-residue peptides, designated EF1-EF6, corresponding to the 6 EF-hand sequences of calbindin D28k, were made. The response of each peptide to Ca2+ addition was assessed by 1H NMR spectroscopy, circular dichroism (CD) spectroscopy, and agarose gel electrophoresis. The Ca2+ binding by CD experiments was performed at two peptide concentrations, 20 and 200 microM, and the NMR studies at peptide concentrations ranging from 20 to 100 microM. The CD and 1H NMR data show that five of the six peptides bind Ca2+ as isolated peptides, namely, EF1, EF3, EF4, EF5, and EF6. The EF6 peptide appears to bind Ca2+ with lower affinity than the other four functional sites. In contrast, EF2 does not appear to bind Ca2+ under any of the spectroscopic conditions tested. The data suggest that at least five of the six putative sites in the native protein bind Ca2+, although their relative affinities cannot be deduced from studies of the isolated peptides.

In EF-hand calcium binding sites of known structure, the side chain provided by the ninth EF-loop position lies at the entrance of the shortest pathway connecting the metal binding cavity to solvent. The location of this residue suggests that it could serve as a "gateway", providing steric and electrostatic control over the kinetics of Ca2+ binding and dissociation. To test this hypothesis, the present study has engineered the putative gateway side chain of a model EF-hand site and determined the resulting effects on metal ion affinity and dissociation kinetics. The model site chosen was that of the Escherichia coli galactose binding protein (GBP), in which the putative gateway is a Gln side chain. Nine engineered GBP molecules were generated and isolated, each exhibiting native-like activity and global conformation. Two control substitutions at the fourth EF-loop position, a noncoordinating surface residue, had no significant effect on either the equilibrium or the kinetics of the model site. The remaining seven proteins, which possessed unique substitutions at the ninth EF-loop position (Glu, Asn, Asp, Thr, Ser, Gly, Ala), in each case significantly altered the affinity or dissociation kinetics of the site for Tb3+, used as a probe metal ion. Neutral side chains at the gateway position yielded a 590-fold range of Tb3+ dissociation kinetics but only a 3-fold range of Tb3+ affinities, indicating that the size or polarity of these substitutions alters the transition state barrier for metal binding and release without substantially shifting the equilibrium. In contrast, acidic side chains yielded as much as a 34-fold decrease in the Tb3+ off-rate and a 33-fold increase in Tb3+ affinity, suggesting that a negative charge at the gateway position increases the equilibrium stability of the bound metal ion and thereby slows metal release. Thus, kinetic tuning by the gateway side chain exhibits both transition state and ground state tuning mechanisms. In natural EF-hand sequences, different gateway side chains are used by slow buffering sites and rapid signaling sites, providing evidence that the gateway position is an important physiological determinant of metal binding kinetics.

Calbindin D28K forms a Ca(2+)-dissociable complex with mellitin in vitro.

Biochem Mol Biol Int. 1996; 38: 1199-210

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Calbindin D28K (CB), a cytosolic calcium binding protein (CBP), forms a macromolecular complex with the polypeptide mellitin (ME) the absence of calcium, which can be reversibly dissociated by the addition of Ca2+. The molar ratio of CB:ME constituted in this complex is 1:4, suggesting that CB interacts with the tetrameric form of ME. Like free tetrameric ME, the CB:ME complex does not migrate into 15% non-denaturing polyacrylamide electrophoretic gels, although both constituents migrate normally after irreversible complex denaturation by heating in sodium dodecyl sulphate (SDS). The interaction of these two proteins can be distinguished from the association of calmodulin (CM) with ME, which forms a reversibly dissociable, equimolar complex in the presence of Ca2+ and a stable non-migrating complex (molar ratio = 1:12) in its absence. Thus, CB and CM appear to bind ME under different Ca2+ regulatory control, suggesting possible roles for CB as a Ca(2+)-dependent regulatory binding protein.

S100B(beta beta), a member of the S100 protein family, is a Ca(2+)-binding protein with noncovalent interactions at its dimer interface. Each apo-S100 beta subunit (91 residues) has four alpha-helices and a small antiparallel beta-sheet, consistent with two predicted helix-loop-helix Ca(2+)-binding domains known as EF-hands [Amburgey et al. (1995) J. Biomol. NMR 6, 171-179]. The three-dimensional solution structure of apo-S100B(beta beta) from rat has been determined using 2672 distance (14.7 per residue) and 88 dihedral angle restraints derived from multidimensional nuclear magnetic resonance spectroscopy. Apo-S100B (beta beta) is found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with its high solubility. At the symmetric dimer interface, 172 intermolecular nuclear Overhauser effect correlations (NOEs) define the antiparallel alignment of helix I with I' and of helix IV with IV'. The perpendicular association of these pairs of antiparallel helices forms an X-type four-helical bundle at the dimer interface. Whereas, the four helices within each apo-S100 beta subunit adopt a unicornate-type four-helix bundle, with helix I protruding from the parallel bundle of helices II, III, and IV. Accordingly, the orientation of helix III relative to helices I, II, and IV in each subunit differs significantly from that known for other Ca(2+)-binding proteins. Indeed, the interhelical angle (omega) observed in the C-terminal EF-hand of apo-S100 beta is -142 degrees, whereas omega ranges from 118 degrees to 145 degrees in the apo state and from 84 degrees to 128 degrees in the Ca(2+)-bound state for the EF-hands of calbindin D9k, calcyclin, and calmodulin. Thus, a significant conformational change in the C-terminal EF-hand would be required for it to adopt a structure typical of the Ca(2+)-bound state, which could readily explain the dramatic spectral effects observed upon the addition of Ca2+ to apo-S100B(beta beta).

Ca2+ regulates muscle contraction by reversible binding to troponin C (TnC), the Ca(2+)-binding subunit of troponin complex. In order to identify acidic amino acids exposed on its surface, carboxyl groups in TnC were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, then labeled with dinitrophenylethylenediamine. Labeled protein was then digested with trypsin and thermolysin, and the resulting peptides were purified by HPLC. Modified peptides were detected by their specific absorbance at 360 nm, and labeled amino acids in these peptides were identified by sequence analysis. Although the total incorporation of label into TnC was only 2.6 mol/mol, we found that 14 of the 46 carboxyl groups of TnC were partially labeled. The labeled carboxyl groups were located in surface regions of the known three-dimensional structure of TnC which may interact with other components of the troponin complex.

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

Interaction of S100a0 protein with the actin capping protein, CapZ: characterization of a putative S100a0 binding site in CapZ alpha-subunit.

Biochem Biophys Res Commun. 1996; 221: 46-50

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S100a0, a Ca2+-binding protein expressed predominantly in cardiac and skeletal muscle tissues, was demonstrated by chemical cross-linking to interact in a Ca2+ -dependent manner with the actin capping protein CapZ. TRTK-12, a peptide contained within the COOH-terminal region of CapZalpha, inhibited S100a0: CapZ interaction in a dose-dependent manner. TRTK-12 was shown by cross-linking to bind S100a0 in the presence of Ca2+, and by fluorescence spectrophotometry to interact in a saturable manner with the anionic phospholipid and a regulator of CapZ activity, phosphatidylinositol 4-monophosphate; but not with the neutral phospholipid, phosphatidylcholine. These data suggest S100a0 and polyphosphoinositides bind to the same COOH-terminal region of CapZalpha, thus potentially modulating CapZ activity.

A model for the solution structure of horse heart ferricytochrome c has been determined by nuclear magnetic resonance spectroscopy combined with hybrid distance geometry-simulated annealing calculations. Forty-four highly refined structures were obtained using a total of 1671 distance constraints based on the observed magnitude of nuclear Overhauser effects and 58 torsion angle restrains based on the magnitude of determined J-coupling constants. The model incorporates six long-lived water molecules detected by pseudo-two-dimensional NOESY-TOCSY spectra. The all-residue root mean square deviation about the average structure is 0.33 +/- 0.04 A for the backbone N, C alpha, and C' atoms and 0.83 +/- 0.05 A for all heavy atoms. The overall topology of the model for solution structure is very similar to that seen in previously reported models for crystal structures of homologous c-type cytochromes though there are a number of significant differences in detailed aspects of the structure. Two of the three main helices display localized irregularities in helical hydrogen bonding resulting in bifurcation of main chain hydrogen bond acceptor carbonyls. The N- and C-terminal helices are tightly packed and display several interhelical interactions not seen in reported crystal models. To provide an independent measure of the accuracy of the model for the oxidized protein, the expected pseudocontact shifts induced by the spin 1/2 iron were compared to the observed redox-dependent chemical shift changes. These comparisons confirm the general accuracy of the model for the oxidized protein and its observed differences with the structure of the reduced protein. The structures of the reduced and oxidized states of the protein provide a template to explain a range of physical and biological data spanning the redox properties, folding, molecular recognition, and stability of the cytochrome c molecule. For example, a redox-dependent reorganization of surface residues at the heme edge can be directly related to the redox behavior of the protein and thereby provides a previously undocumented linkage between structural change potentially associated with molecular recognition of redox partners and the fundamental parameters governing electron transfer.

Introduction of a fifth carboxylate ligand heightens the affinity of the oncomodulin CD and EF sites for Ca2+.

Biochemistry. 1996; 35: 5856-69

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The acid-pair hypothesis, proposed by Reid and Hodges [(1980) J. Theor. Biol. 84, 401-444], suggests that the affinity of an EF-hand motif for Ca2+ will be maximal with four acidic ligands, paired along the +x, -x and +z, -z axes. Addition of a fifth anionic ligand is predicted to reduce Ca(2+)-binding affinity, as a consequence of increased electrostatic repulsion. Interestingly, for oncomodulin, we observe that introduction of a fifth carboxylate residue at the +z position in the CD coordination sphere or at the -x position in the EF coordination sphere significantly increases the affinity of those sites for Ca2+. The variants resulting from replacement of serine-55 by aspartate (S55D), glycine-98 by aspartate (G98D), and the combined mutations (55/98) have been examined in Ca(2+)- and Mg(2+)-binding studies, titration calorimetry, and differential scanning calorimetry. The KCa for the CD site is reduced from 800 to 67 nM by the S55D mutation, while KCa for the EF site is reduced from 45 to 4 nM by the G98D mutation. Both mutations destabilize the apo form of the protein and increase the thermal stability on the Ca(2+)-bound state. Interestingly, the S55D mutation also increases the affinity of the oncomodulin CD site for Mg2+, decreasing the dissociation constant from > 1 mM to approximately 30 microM. This increase in affinity is reflected in a substantially increased thermal stability of the Mg(2+)-bound form of the protein. In 0.15 M NaCl, 0.025 M Hepes (pH 7.4), and 0.01 M Mg2+, the wild-type protein denatures at 68.5 degrees C. By contrast, under identical conditions, the S55D mutations denatures at 79.0 degrees C. The increased metal ion-binding affinity displayed by the variant proteins may result in part from preferential destabilization of the apo-protein by the additional carboxylate.

The C-terminal domain from the muscle protein troponin C (TnC) comprises two helix-loop-helix calcium-binding sites (residues 90-162). The assembly of these two sites is governed by calcium binding enabling a synthetic C-terminal domain to be preferentially and stoichiometrically assembled from two synthetic peptides (residues 93-126, SCIII, and 129-162, SCIV) in the presence of calcium only. It is therefore of great interest to know how closely the structure of this heterodimeric domain is to the intact protein domain. Analysis of such a structure has important implications in protein engineering and in understanding the stability of calcium-binding proteins in terms of biological function. The solution structure of this heterodimeric protein was determined by 1H NMR spectroscopy using 802 NOE derived distance restraints and 23 phi and 22 chi angle restraints. Distance geometry-simulated annealing calculations yielded a family of 42 converged structures (rmsd 0.86 +/- 0.17 A) showing an arrangement of four alpha-helices similar in fold to the C-terminal of troponin C. The dimer interface has several important interactions between helix pairs E/H and F/G responsible for the association of the two peptides. However, neither the peptide complex nor the solution NMR structure of TnC pack as tightly as that observed in the TnC X-ray structure. The interhelical distance between the F/G helix is about 1.4 A greater in solution than in the crystal. A comparison of the exposed surface area of the hydrophobic residues in the SCIII/SCIV heterodimer revealed that residues 1104, Y112, and 1121 are more exposed than in the previously determined solution structure of the SCIII homodimer. These residues are important for the interaction with the inhibitory region of TnI and provide evidence for their involvement in the regulation of muscle contraction.

The homodimeric S100 protein calcyclin has been studied in the apo state by two-dimensional 1H NMR spectroscopy. Using a combination of scalar correlation and NOE experiments, sequence-specific 1H NMR assignments were obtained for all but one backbone and > 90% of the side-chain resonances. To our knowledge, the 2 x 90 residue (20 kDa) calcyclin dimer is the largest protein system for which such complete assignments have been made by purely homonuclear methods. Sequential and medium-range NOEs and slowly exchanging backbone amide protons identified directly the four helices and the short antiparallel beta-type interaction between the two binding loops that comprise each subunit of the dimer. Further analysis of NOEs enabled the unambiguous assignment of 556 intrasubunit distance constraints, 24 intrasubunit hydrogen bonding constraints, and 2 x 26 intersubunit distance constraints. The conformation of the monomer subunit was refined by distance geometry and restrained molecular dynamics calculations using the intrasubunit constraints only. Calculation of the dimer structure starting from this conformational ensemble has been reported elsewhere. The extent of structural homology among the apo calcyclin subunit, the monomer subunit of apo S100 beta, and monomeric apo calbindin D9k has been examined in detail by comparing 1H NMR chemical shifts and secondary structures. This analysis was extended to a comprehensive comparison of the three-dimensional structures of the calcyclin monomer subunit and calbindin D9k, which revealed greater similarity in the packing of their hydrophobic cores than was anticipated previously. Together, these results support the hypothesis that all members of the S100 family have similar core structures and similar modes of dimerization. Analysis of the amphiphilicity of Helix IV is used to explain why calbindin D9k is monomeric, but full-length S100 proteins form homodimers.

The EF-hand is a highly conserved Ca(2+)-binding motif found in many cytosolic Ca(2+)-modulated proteins. Here we report the crystal structure at 2.0 A resolution of the carboxy-terminal domain of human BM-40 (SPARC, osteonectin), an extracellular matrix protein containing an EF-hand pair. The two EF-hands interact canonically but their detailed structures are unusual. In the first EF-hand a one-residue insertion is accommodated by a cis-peptide bond and by substituting a carboxylate by a peptide carbonyl as a Ca2+ ligand. The second EF-hand is stabilized by a disulphide bond. The EF-hand pair interacts tightly with an amphiphilic amino-terminal helix, reminiscent of target peptide binding by calmodulin. The present structure defines a novel protein module occurring in several other extracellular proteins.

Antibody recognition of calcium-binding proteins depends on their calcium-binding status.

J Neurochem. 1996; 66: 764-71

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Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.

BACKGROUND & AIMS: Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. METHODS: Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. RESULTS: CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. CONCLUSIONS: These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

Calcium binding to the regulatory N-domain of skeletal muscle troponin C occurs in a stepwise manner.

Biochemistry. 1995; 34: 8330-40

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Ca2+ binding to a recombinant regulatory N-domain (residues 1-90) of chicken troponin C (NTnC) has been investigated with the use of heteronuclear multidimensional NMR spectroscopy. The protein has been cloned in pET3a vector and expressed in minimal media in Escherichia coli to allow uniform 15N and 13C labeling. The NMR spectra have been resolved and completely assigned [Gagne et al. (1994) Protein Sci. 3, 1961-1974]. Ca2+ titration monitored by 2D (1H, 15N)-HMQC NMR spectral changes revealed that Ca2+ binding to sites I and II of NTnC is a stepwise process and that chemical shift changes occur throughout the N-domain upon the binding of each Ca2+. The Ca2+ dissociation constants for the binding of the first and second Ca2+ were determined to be 0.8 microM < or = Kd1 < or = 3 microM and 5 microM < or = Kd2 < or = 23 microM, respectively. This mechanism is believed to represent that of the N-domain in intact TnC since we have shown earlier that the properties of the N-domain (1-90) were identical to those of the N-domain in intact TnC [Li et al. (1994) Biochemistry 33, 917-925]. In contrast, however, our previous Ca2+ fluorescence and far-UV CD studies on F29W NTnC and F29W TnC indicated cooperative Ca2+ binding to sites I/II and no detectable differences in their affinities. To rationalize these observations, a direct comparison was made of the Ca2+ titration of NTnC and F29W NTnC as monitored by far-UV CD spectroscopy. Unlike F29W NTnC, NTnC gave a biphasic curve with binding constants in reasonable agreement with the NMR data. Although the far-UV CD spectra of NTnC and the F29W NTnC domain were the same in the absence of Ca2+, the Ca(2+)-induced negative ellipticity increase for NTnC is significantly smaller than for F29W NTnC. These observations indicate that the F29W mutation has perturbed the Ca2+ binding properties of the N-domain and its CD spectroscopic properties in the Ca(2+)-saturated state.

Calcium binding proteins are subdivided into two major families: the EF-family and the Annexin family. The EF-hand family is distinguished by the characteristic helix-loop-helix motif which consists of two alpha-helices separated by a loop. The EF-hand Ca2+(-)binding protein family contain subfamilies rich in proline, glutamic acid, serine and threonine residues (called PEST sequences) and non PEST-containing subfamilies. A few of the Ca2+(-)binding proteins contain KFERQ-like sequences which are thought to be possible signals for lysosomal degradation. Arginine pairs (RR) which have also been suggested to act as signals for proteolysis were found to be few or absent. However, most EF-hand proteins were found to possess lysine pairs (KK) which may also act as signals for proteolysis.

A new self-correcting distance geometry method for predicting the three-dimensional structure of small globular proteins was assessed with a test set of 8 helical proteins. With the knowledge of the amino acid sequence and the helical segments, our completely automated method calculated the correct backbone topology of six proteins. The accuracy of the predicted structures ranged from 2.3 A to 3.1 A for the helical segments compared to the experimentally determined structures. For two proteins, the predicted constraints were not restrictive enough to yield a conclusive prediction. The method can be applied to all small globular proteins, provided the secondary structure is known from NMR analysis or can be predicted with high reliability.

Cooperativity, the ability of ligand binding at one site on a macromolecule to influence ligand binding at a different site on the same macromolecule, is a fascinating biological property that is often poorly explained in textbooks. The Hill coefficient is commonly used in biophysical studies of cooperative systems although it is not a quantitative measure of cooperativity. The free energy of interaction between binding sites (delta delta G) is a more stringent definition of cooperativity and provides a direct quantitative measure of how the binding of ligand at one site affects the ligand affinity of another site.

An ELISA for grancalcin, a novel cytosolic calcium-binding protein present in leukocytes.

J Immunol Methods. 1995; 185: 1-8

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Grancalcin is a newly discovered cytosolic calcium-binding protein, belonging to the group of EF-hand proteins. Grancalcin is specifically associated with cells originating in the bone marrow. Grancalcin binds reversibly to secretory vesicles and plasma membranes in human neutrophils and might therefore play a role in the regulation of vesicle/granule exocytosis. We describe here the production of recombinant grancalcin, the generation of antibodies to the protein and the development of a specific, accurate and sensitive ELISA for the detection of human leukocytic grancalcin. This ELISA may be useful for monitoring leukocyte infiltration into tissues.

The three-dimensional structure of chemotactic Che Y protein from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. A total of 20 converged structures were computed from 1545 conformationally relevant distance restraints derived from 1858 unambiguously assigned NOE cross-correlations. The resulting average pairwise root-mean-square deviation is 1.03 A for the backbone atoms and 1.69 A for all heavy atoms. If residues in the regions structurally least defined (1 to 5, 47 to 50, 76 to 79, 88 to 91 and 124 to 129) are excluded from the analysis, the root-mean-square deviations are reduced to 0.53 A and 1.23 A, respectively. The solution structure is closely similar to the refined X-ray crystal structure, except in the regions found to be less defined by NMR spectroscopy. The root-mean-square deviation between the average solution structure and the X-ray crystal structure is 0.92 A for the backbone residues (2 to 129). The highly refined solution structure determined herewith provides an essential background to delineate functionally important conformational changes brought about by different effectors.

This chapter describes a current perception of the molecular interactions regulating myofilament activity in heart cells. The focus is on the interaction between troponin-C (TnC), the Ca(2+)-receptor and troponin I (TnI), an inhibitory protein. It is this interaction that appears to form a molecular switch that turns on the thin filament. It will be seen that control of the actin-myosin reaction is not only through Ca(2+)-binding to TnC, but also through steric, cooperative and allosteric processes involving all of the main myofilament proteins-actin, myosin, tropomyosin (Tm), troponin T (TnT), TnC, and TnI. The process is modulated by covalent and non-covalent mechanisms. The process is altered in diverse myopathies and pathologies of the heart and is a target for pharmacological manipulation by a new class of inotropic agents, the "Ca(2+)-sensitizers".

Recently, we described the isolation of a mouse cDNA clone encoding a novel Ca(2+)-binding protein, tentatively designated as reticulocalbin [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Reticulocalbin is a lumenal protein of the endoplasmic reticulum (ER) with a molecular weight of 44,000 and has six repeats of a domain containing the high affinity EF-hand Ca(2+)-binding motif. The protein has the sequence, His-Asp-Glu-Leu (HDEL), at its carboxy terminus, which serves as a signal for its retention in the ER of cells. To examine the importance of the putative Ca(2+)-binding domains as well as the carboxy-terminal HDEL sequence, we have cloned the human homologue of reticulocalbin. The sequence of this clone revealed a novel protein with 95% identity in amino acid sequence to the mouse reticulocalbin, indicating that this molecule has been evolutionarily conserved in mammals. As was found for the mouse reticulocalbin, the human homologue showed six repeats of a domain containing EF-hand motifs. Interestingly, conservation of the amino acid sequence was not restricted to the Ca(2+)-binding motifs, consistent with the possibility that reticulocalbin plays some role(s) besides Ca(2+)-binding. As was found for the mouse homologue, the protein has the HDEL sequence at its carboxy terminus instead of the Lys-Asp-Glu-Leu (KDEL) sequence, which is more common as a signal for the retention of resident proteins in the ER of animal cells. The conservation of the HDEL sequence in reticulocalbin in both species raises the possibility that this sequence has some roles in the function(s) of this protein family.

Conformational changes and calcium binding by calretinin and its recombinant fragments containing different sets of EF hand motifs.

Biochemistry. 1995; 34: 15389-94

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Four recombinant fragments, representing different sets of EF-hand motifs of rat calretinin (CR) (I-II, I-III, III-VI, IV-VI), were prepared, and their Ca2+ -induced conformational changes were compared with those of full-length recombinant CR. All fragments were able to bind calcium ions as shown by 45Ca2+ overlay method on nitrocellulose and fluorescence measurements. The intrinsic tryptophan fluorescence intensity (FI) of apo-CR reversibly increased about 3-fold upon addition of calcium, indicating a change of conformation. The FI of fragments I-II (Trp 25) and I-III (Trp 25 and 116) increased about 1.4-fold on calcium binding, but that of fragment III-VI (Trp 116) increased 3.5-fold. Calcium titration of CR monitored by Trp fluorescence intensity showed that recombinant CR and some fragments bound Ca2+ with high affinity (Kd below 0.4 microM) and with high cooperativity. An apparent Hill coefficient for Ca2+ -induced fluorescence changes in CR was about 3.7. CR bound to organomercurial-agarose Cys 101 and 266 did not form cystine. The fluorescence intensities of cysteine-linked fluorescent probes 5-iodoacetamidofluorescein and N-(1-pyreneiodoacetamide) were increased approximately 1.3-fold upon calcium binding by CR. These data indicate that CR binds Ca2+ with high affinity and cooperativity and that this binding induces a change of conformation that involves the interaction of different parts of the molecule. Taken together, our results suggest that CR works as an on/off switch within a narrow range of free Ca2+ by interacting with as yet unidentified targets.

Preference of calcium-dependent interactions between calmodulin-like domains of calpain and calpastatin subdomains.

FEBS Lett. 1995; 362: 93-7

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Calpastatin molecule contains four repeated inhibition domains, each having highly conserved internal regions A, B and C. The synthetic oligopeptides of regions A and C had no calpain inhibition activity while region B oligopeptide showed weak inhibition activity. Real-time biomolecular interaction analysis using a BIAcore instrument revealed that the bacterially expressed calmodulin-like domain of the calpain large subunit (L-CaMLD) and that of the small subunit (S-CaMLD) interacted, in a Ca(2+)-dependent fashion, preferentially with the immobilized synthetic oligopeptide of region A and that of region C, respectively. Calmodulin showed no specific binding to these oligopeptides. The tripartite structure of the calpastatin functional domain may confer the specific interactions with the protease domain and the two CaMLDs of calpain.

Recoverin is an N-myristoylated calcium-binding protein present in the photoreceptor cells of the mammalian retina. It is believed to function as a calcium sensor in visual signal transduction by coupling the kinetics of the recovery phase of the photoresponse to changes in the levels of intracellular Ca2+. Upon binding Ca2+, recoverin undergoes a conformational change that allows it to associate with membranes in a manner that requires N-myristoyl modification. It has been proposed that, in the Ca(2+)-free conformation, the myristoyl group is sequestered in a hydrophobic part of the protein, and in the Ca(2+)-bound conformation, the myristoyl group is exposed to solution. The crystal structure of Ca(2+)-bound recoverin reveals an exposed cluster of hydrophobic residues, raising the possibility that residues in this region may function as part of an intramolecular myristoyl binding site. Fluorescence spectroscopy analysis of interactions between recoverin and 1-anilinonaphthalene-8-sulfonate (ANS) shows that an increase in solvent-accessible hydrophobic surface accompanies Ca2+ binding. 1H nuclear magnetic resonance (NMR) spectra of myristoyl protons show dispersed chemical shifts in the Ca(2+)-free conformation that become relatively uniform upon the addition of Ca2+. Two-dimensional nuclear Overhauser effect (NOE) spectra of Ca(2+)-free recoverin show NOE contacts between myristoyl protons and aromatic ring protons. Tryptophan fluorescence quenching by acrylamide indicates that the myristoyl group is in proximity to a tryptophan residue only in the Ca(2+)-free conformation. These results indicate that the myristoyl group is in contact with residues in the hydrophobic cluster in Ca(2+)-free recoverin and that it is exposed to solution in the Ca(2+)-bound conformation.

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C.

Protein Sci. 1995; 4: 671-80

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The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.

The solution structure of Cucurbita maxima trypsin inhibitor-V (CMTI-V), which is also a specific inhibitor of the blood coagulation protein, factor XIIa, was determined by 1H NMR spectroscopy in combination with a distance-geometry and simulated annealing algorithm. Sequence-specific resonance assignments were made for all the main-chain and most of the side-chain hydrogens. Stereospecific assignments were also made for some of the beta-, gamma-, delta-, and epsilon-hydrogens and valine methyl hydrogens. The ring conformations of all six prolines in the inhibitor were determined on the basis of 1H-1H vicinal coupling constant patterns; most of the proline ring hydrogens were stereospecifically assigned on the basis of vicinal coupling constant and intraresidue nuclear Overhauser effect (NOE) patterns. Distance constraints were determined on the basis of NOEs between pairs of hydrogens. Dihedral angle constraints were determined from estimates of scalar coupling constants and intraresidue NOEs. On the basis of 727 interproton distance and 111 torsion angle constraints, which included backbone phi angles and side-chain chi 1, chi 2, chi 3, and chi 4 angles, 22 structures were calculated by a distance geometry algorithm and refined by energy minimization and simulated annealing methods. Both main-chain and side-chain atoms are well-defined, except for a loop region, two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 22 individual structures and the mean structure obtained by averaging their coordinates is 0.58 +/- 0.06 A for the main-chain atoms and 1.01 +/- 0.07 A for all the non-hydrogen atoms of residues 3-40 and 49-67. These structures were compared to the X-ray crystallographic structure of another protein of the same inhibitor family-chymotrypsin inhibitor-2 from barley seeds [CI-2; McPhalen, C. A., & James, M. N. G. (1987) Biochemistry 26, 261-269]. The main-chain folding patterns are highly similar for the two proteins, which possess 62% sequence differences. However, major differences are noted in the N- and C-terminal segments, which may be due to the presence of a disulfide bridge in CMTI-V, but not in CI-2.

Human S100b protein: formation of a tetramer from synthetic calcium-binding site peptides.

Protein Sci. 1995; 4: 765-72

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Human brain S100b protein is a unique calcium-binding protein comprised of two identical 91-amino acid polypeptide chains that each contain two proposed helix-loop-helix (EF-hand) calcium-binding sites. In order to probe the assembly of the four calcium-binding sites in S100b, a peptide comprised of the N-terminal 46 residues of S100b protein was synthesized and studied by CD and 1H NMR spectroscopies as a function of concentration and temperature. At relatively high peptide concentrations and in the absence of calcium, the peptide exhibited a significant proportion of alpha-helix (45%). Decreasing the peptide concentration led to a loss of alpha-helix as monitored by CD spectroscopy and coincident changes in the 1H NMR spectrum. These changes were also observed by 1H NMR spectroscopy as a function of temperature where it was observed that the Tm of the peptide was lowered approximately 14 degrees C with a 17-fold decrease in peptide concentration. Sedimentation equilibrium studies were used to determine that the peptide formed a tetramer in solution in the absence of calcium. It is proposed that this tetrameric fold also occurs in S100b and is a result of the interaction of portions of all four calcium-binding sites.

Cardiac troponin I induced conformational changes in cardiac troponin C as monitored by NMR using site-directed spin and isotope labeling.

Biochemistry. 1995; 34: 13343-52

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Conformational changes in both free cardiac troponin C (cTnC) and in complex with a recombinant troponin I protein [cTnI(33-211), cTnI(33-80), or cTnI (86-211)] were observed by means of a combination of selective carbon-13 and spin labeling. The paramagnetic effect from the nitroxide spin label, MTSSL, attached to cTnC(C35S) at Cys 84 allowed measurement of the relative distances to the 13C-methyl groups of the 10 methionines of cTnC in the monomer or complex. All 10 1H-13C correlations in the heteronuclear single- and multiple-quantum coherence (HSMQC) spectrum of [13C-methyl] Met cTnC in the complex with cTnI(33-211) were previously assigned [Krudy, G. A., Kleerekoper, Q., Guo, X., Howarth, J. W., Solaro, R. J., & Rosevear, P. R. (1994) J. Biol. Chem. 269, 23731-23735]. In the presence of oxidized spin label, nine of the 10 Met methyl 1H-13C correlations of cTnC were significantly broadened in the cTnC(C35S) monomer. This suggests flexibility within the central helix, or interdomain D/E helical linker, bringing the N- and C-terminal domains in closer proximity than predicted from the crystallographic structure of TnC. In the spin-labeled cTnC(C35S). cTnI(33-211) complex only N-terminal Met methyl 1H-13C correlations of cTnC(C35S) were paramagnetically broadened beyond detection, whereas correlations for Met residues (103, 120, 137, and 157) in the C-terminal domain were not. Thus, complex formation with cTnI decreases interdomain flexibility and maintains cTnC in an extended conformation. This agrees with the recently published study suggesting that sTnC is extended when bound to sTnI [Olah, G. A., & Trewhella, J. (1994) Biochemistry 33, 12800-12806]. The recombinant N-terminal domain of cTnI, cTnI(33-80), gave similar results as observed with cTnI(33-211) when complexed with spin-labeled cTnC(C35S). However, complex formation with the C-terminal fragment, cTnI(86-211), which contains the inhibitory sequence, is insufficient to maintain cTnC extended to the amount observed with either cTnI(33-211) or cTnI(33-80); although compared to that observed in free cTnC, it does cause decreased flexibility in the interdomain linker. In the absence of the N-terminal domain of cTnI, there is a decrease in flexibility within the N-terminal domain of cTnC. Interestingly, the N-terminal domain of cTnC in the reduced spin-labeled complex with cTnI(86-211), in the presence of ascorbate, showed two distinct conformations which were not seen in the complex with cTnI(33-211).(ABSTRACT TRUNCATED AT 400 WORDS)

Calbindin D28K binds 3 mol of terbium per mol of protein. To determine which of six EF-hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF-hand 2 (RCaBP delta 2), the other lacking EF-hands 2 and 6 (RCaBP delta 2,6), and the third containing only EF-hands 3 and 4 (RCaBP delta 1,2,5,6), and examined their binding properties by fluorescence techniques. Full-length calbindin D28K and RCaBP delta 2 and RCaBP delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EF-hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C. Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites. To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a delta 1,2,5,6 RCaBP construct. The truncated protein RCaBP delta 1,2,5,6 contained a single terbium-binding site. Analysis of the terbium binding to RCaBP delta 1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D28K are responsible for terbium binding.

Characterization of the N-terminal half-saturated state of calbindin D9k: NMR studies of the N56A mutant.

Protein Sci. 1995; 4: 1045-55

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Calbindin D9k is a small EF-hand protein that binds two calcium ions with positive cooperativity. The molecular basis of cooperativity for the binding pathway where the first ion binds in the N-terminal site (1) is investigated by NMR experiments on the half-saturated state of the N56A mutant, which exhibits sequential yet cooperative binding (Linse S, Chazin WJ, 1995, Protein Sci 4:1038-1044). Analysis of calcium-induced changes in chemical shifts, amide proton exchange rates, and NOEs indicates that ion binding to the N-terminal binding loop causes significant changes in conformation and/or dynamics throughout the protein. In particular, all three parameters indicate that the hydrophobic core undergoes a change in packing to a conformation very similar to the calcium-loaded state. These results are similar to those observed for the (Cd2+)1 state of the wild-type protein, a model for the complementary half-saturated state with an ion bound in the C-terminal site (II). Thus, with respect to cooperativity in either of the binding pathways, binding of the first ion drives the conformation and dynamics of the protein far toward the (Ca2+)2 state, thereby facilitating binding of the second ion. Comparison with the half-saturated state of the analogous E65Q mutant confirms that mutation of this critical bidentate calcium ligand at position 12 of the consensus EF-hand binding loop causes very significant structural perturbations. This result has important implications regarding numerous studies that have utilized mutation of this critical residue for site deactivation.

The concentration of protein in a solution has been found to have a significant effect on ion binding affinity. It is well known that an increase in ionic strength of the solvent medium by addition of salt modulates the ion-binding affinity of a charged protein due to electrostatic screening. In recent Monte Carlo simulations, a similar screening has been detected to arise from an increase in the concentration of the protein itself. Experimental results are presented here that verify the theoretical predictions; high concentrations of the negatively charged proteins calbindin D9k and calmodulin are found to reduce their affinity for divalent cations. The Ca(2+)-binding constant of the C-terminal site in the Asn-56 --> Ala mutant of calbindin D9k has been measured at seven different protein concentrations ranging from 27 microM to 7.35 mM by using 1H NMR. A 94% reduction in affinity is observed when going from the lowest to the highest protein concentration. For calmodulin, we have measured the average Mg(2+)-binding constant of sites I and II at 0.325, 1.08, and 3.25 mM protein and find a 13-fold difference between the two extremes. Monte Carlo calculations have been performed for the two cases described above to provide a direct comparison of the experimental and simulated effects of protein concentration on metal ion affinities. The overall agreement between theory and experiment is good. The results have important implications for all biological systems involving interactions between charged species.

Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.

Protein Sci. 1995; 4: 1038-44

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Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k.

S-100 beta, a calcium binding protein produced by astrocytes, has been proposed to be a neuronotropic agent. In order to test the tropic effects of S-100 beta in vivo, the technique of cell transplantation was used. C6 glioma cells and C6 cells containing a S-100 beta antisense gene (C6AS) were transplanted into contralateral hippocampi. 5-HT immunoreactive, varicose fibers with a normal appearance penetrated into the glioma mass and were seen in high density around the C6 cell mass. However, 5-HT fibers with enlarged, abnormal varicosities were seen bordering C6AS tissue and were very rarely observed within the C6AS cell mass. Extracellular S-100 beta from normal C6 cells may function as a growth factor on sprouting serotonergic fibers.

The solution structure of the glucocorticoid receptor (GR) DNA-binding domain (DBD), consisting of 93 residues, has been refined from two and three-dimensional NMR data using an ensemble iterative relaxation matrix approach followed by direct NOE refinement with DINOSAUR. A set of 47 structures of the rat GR fragment Cys440-Arg510 was generated with distance geometry and further refined with a combination of restrained energy minimization and restrained molecular dynamics in a parallel refinement protocol. Distance constraints were obtained from an extensive set of NOE build-up curves in H2O and 2H2O via relaxation matrix calculations (1186 distance constraints from NOE intensities, 10 phi and 22 chi 1 dihedral angle constraints from J- coupling data were used for the calculations). The root-mean-square deviation values of the 11 best structures on the well-determined part of the protein (Cys440 to Ser448, His451 to Glu469 and Pro493 to Glu508) are 0.60 A and 1.20 A from the average for backbone and all heavy atoms, respectively. The final structures have R-factors around 0.40 and good stereochemical qualities. The first zinc-coordinating domain of the GR DBD is very similar to the crystal structure with a root-mean-square difference of 1.4 A. The second zinc-coordinating domain is still disordered in solution. No secondary structure element is found in this domain in the free state. As suggested by crystallographic studies on the estrogen receptor DBD-DNA and GR DBD-DNA complexes, part of this region will form a distorted helix and the D-box will undergo a conformational change upon cooperative binding to DNA.

We have designed a distance geometry-based method for obtaining the tertiary fold of a protein from a limited number of structure-specific distance restraints and the secondary structure assignment. Interresidue distances were predicted from patterns of conserved hydrophobic amino acids deduced from multiple alignments. A simple model chain representing the protein was then folded by projecting its distance matrix into Euclidean spaces with gradually decreasing dimensionality until a final three-dimensional embedding was achieved. Tangled conformations produced by the projection steps were eliminated using a novel filtering algorithm. Information on various aspects of protein structure such as accessibility and chirality was incorporated into the conformation refinement, increasing the robustness of the algorithm. The method successfully identified the correct folds of three small proteins from a small number of restraints, indicating that it could serve as a useful computational tool in protein structure determination from NMR data.

Short amino acid sequences that interact with the Ca2+ binding protein S-100b were identified by screening a bacteriophage random peptide display library. S-100b binding bacteriophages were selected by Ca(2+)-dependent affinity chromatography, and the sequence of the random peptide insert contained in 51 clones was determined. Alignment of the sequence of 44 unique S-100b binding peptides identified a common motif of eight amino acids. A subgroup of peptides that contained sequences with the highest degree of similarity had the consensus motif (K/R)(L/I)XWXXIL, in which predominantly P, S, and N were found in position 3, and S and D were found in position 5. Analysis of sequence databanks identified a similar sequence in the COOH-terminal region of the alpha-subunit of actin capping proteins. The peptide TRTKIDWNKILS (TRTK-12), corresponding to the region of greatest homology within this region of the subunit of actin capping proteins (e.g. amino acids 265-276 in CapZ alpha 1 and CapZ alpha 2), was synthesized and shown by fluorescence spectrophotometry to bind S-100b in a Ca(2+)-dependent manner. Gel overlay and cross-linking experiments demonstrated the interaction of S-100b with CapZ to be Ca2+ dependent. Moreover, this interaction was blocked by addition of TRTK-12 peptide. These results identify Ca(2+)-dependent S-100b target sequence epitopes and designate the carboxyl terminus of the alpha-subunit of actin capping proteins, like CapZ, to be a target of S-100b activity. The high level of conservation within this region of actin capping proteins and the apparent high affinity of this interaction strongly suggest that the interaction between S-100b and the alpha-subunit of actin capping proteins is biologically significant.

Isolation and characterization of a calmodulin-like protein from Halobacterium salinarium.

J Bacteriol. 1995; 177: 864-6

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The first evidence for a calmodulin-like protein in an archaeon, Halobacterium salinarium, is reported here. The calmodulin-like protein, with a molecular mass of 24 kDa and an estimated pI of 4.8, stimulated cyclic nucleotide phosphodiesterase in a calcium-dependent manner. This stimulation could be suppressed by calmodulin inhibitors. The Ca(2+)-binding ability was verified by 45Ca autoradiography.

A novel EF-hand Ca(2+)-binding protein from abdominal muscle of crustaceans with similarity to calcyphosine from dog thyroidea.

Eur J Biochem. 1995; 227: 97-101

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The amino acid sequence of a novel EF-hand Ca(2+)-binding protein from the abdominal muscle of the crayfish, Orconectes limosus, has been elucidated by tandem mass spectrometry and automated Edman degradation. The name CCBP-23 (23-kDa crustacean Ca(2+)-binding protein) is proposed. The protein can also exist as a disulfide-linked homodimer. The sequence of the monomeric form spans 200 residues with an acetylated N-terminal Ser and reveals four EF-hand domains. The 174-mass-unit difference between the calculated average molecular mass of 22,669.6 Da deduced from the sequence and the obtained electrospray ionization mass spectroscopy (ESI-MS) mass of 22,844 Da has not yet been explained. Partial sequence analysis (137 residues) of CCBP-23 from the lobster, Homarus americanus, showed a sequence identity of 74% with the crayfish protein. Homology searches revealed a 44% sequence identity of CCBP-23 from crayfish to calcyphosine, a Ca(2+)-binding protein from dog thyroidea (Lefort et al., 1989). Although CCBP-23 also shows a 44% identity to R2D5 antigen (Nemoto et al., 1993), we believe that both proteins represent two distinct subgroups within the family of EF-hand proteins.

The three-dimensional solution structure of toxin FS2, a 60-residue polypeptide isolated from the venom of black mamba snake (Dendroaspis polylepis polylepis), has been determined by nuclear magnetic resonance spectroscopy. Using 600 NOE constraints and 55 dihedral angle constraints, a set of 20 structures obtained from distance-geometry calculations was further refined by molecular dynamics calculations using a combined simulated annealing-restrained MD protocol. The resulting 20 conformers, taken to represent the solution structure, give an average rmsd of 1.2 A for their backbone atoms, relative to the average structure. The overall resulting three-fingered structure is similar to those already observed in several postsynaptic neurotoxins, cardiotoxins, and fasciculins, which all share with toxin FS2 the same network of four disulfide bridges. The overall concavity of the molecule, considered as a flat bottomed dish, is oriented toward the C-terminal loop of the molecule. This orientation is similar to that of fasciculins and cardiotoxins but opposite to that of neurotoxins. On the basis of the local rms displacements between the 20 conformers, the structure of the first loop appears to be less well defined in FS2 than in the previously reported neurotoxin structures, but fasciculin 1 shows a similar trend with particularly high temperature factors for this part of the X-ray structure. The concave side which presents most of the positively charged residues is quite similar in FS2 and fasciculin 1. The main difference is shown by the convex side of the third loop, mostly hydrophobic in FS2, in contrast to the pair of negatively charged aspartates in fasciculin 1. This difference could be one of the factors leading to the distinct pharmacological properties-L-type calcium channel blocker for FS2 and cholinesterase inhibitor for fasciculin--observed for these two subgroups of the "angusticeps-type" toxins.

Purification and cation binding properties of the recombinant human S100 calcium-binding protein A3, an EF-hand motif protein with high affinity for zinc.

J Biol Chem. 1995; 270: 21056-61

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The calcium-binding protein S100A3 is an unusual member of the S100 family, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitro with a subunit molecular weight of 11,712. The Zn(2+)-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn2+ per monomer with a [Zn2+]0.5 of 11 microM and a Hill coefficient of 1.4 at physiological ionic strength. The affinity for Ca2+ is too low to be determined by direct methods (KCa > 10 mM). Ca(2+)- and Zn(2+)-binding can be followed by optical methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn2+ and Ca2+, but not of Mg2+, leads to a 4-fold quenching. Ca2+ and Zn2+ promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotometry. Fluorescence titrations with Zn2+ confirmed that there is one set of high affinity binding sites with a [Zn2+]0.5 of 8 microM and a Hill coefficient of 1.3. Binding of Zn2+ to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca2+ confirmed the very low affinity of S100A3 for this ion with a [Ca2+]0.5 of 30 mM and slight negative cooperativity. Mg2+ has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5'-dithiobis(2-nitro-benzoic acid); they display a high reactivity in the metal-free and Ca2+ form, but a 20-fold lowered reactivity in the Zn2+ form of S100A3. Ca(2+)-binding promotes the formation of a solvent-accessible hydrophobic surface as monitored by the 60-fold fluorescence increase of 2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn2+ has no noticeable influence. Our data indicate that Ca2+ and Zn2+ do not bind to the same sites and that under physiological conditions S100A3 is a Zn(2+)-binding rather than a Ca(2+)-binding protein; nevertheless, very specific conformational changes are introduced by either Ca2+ or Zn2+. Since no Zn(2+)-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn(2+)-binding motif.

The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC.TnI.TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25-kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less TnI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.

Investigation of the structural requirements of the troponin C central helix for function.

Biochemistry. 1995; 34: 16789-96

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The two globular Ca(2+)-binding domains of troponin C are connected by a three-turn, exposed central helix. The requirements of this helical linker for regulatory function are not fully understood. In the present work we investigated the structural requirement of the linker using a series of insertion mutations that differ in predicted flexibility. TnCinrc has a nine-residue flexible random coil insert, TnCinpp has a nine-residue rigid polyproline insert (three turns), and TnCin alpha h has a seven-residue insert with high potential of forming alpha-helix. TnCinrc and TnCinpp were defective in the activation of the regulated actomyosin ATPase activity in the presence of Ca2+ when compared to wild type or TnCin alpha h, suggesting that altering the flexibility of the central helix impairs the regulatory function of troponin C. TnCin alpha h, TnCinrc, and TnCinpp had 87% +/- 3, 62% +/- 3, and 58% +/- 2 of the wild type activity, respectively (n = 6). All insertions in the central helix resulted in elongation of molecule compared to wild type TnC as determined by Stokes' radius. The Ca(2+)-affinity, the Ca(2+)-dependence of the actomyosin ATPase, and the stability of the insertion mutants were similar to wild type. Deletions of up to two turns of the central helix have little effect on troponin C function [Dobrowolski, Z., Xu, G-Q., & Hitchcock-DeGregori, S. E. (1991) J. Biol. Chem. 266, 5703-5710]. In another mutant (TnCd11) the entire central helix, 87KEDAKGKSEEE97, was deleted. With TnCd11, activation of the actomyosin ATPase activity in the presence of Ca2+ was normal, but inhibition in the absence of Ca2+ was less effective. Interaction of TnCd11 with TnI was altered. There was a 2-fold excess of TnCd11 in reconstituted Tn complex, consistent with another report [Babu, A., Rao, V. G, Su, H., & Gulati, J. (1993) J. Biol. Chem. 268, 19232-19238]. Our results suggest that the native length and structure of the central helix are optimal for normal regulatory function and that connectivity alone is insufficient for TnC function.

Troponin C (TnC) is an 18 kDa (162-residue) thin-filament calcium-binding protein responsible for triggering muscle contraction upon the release of calcium from the sarcoplasmic reticulum. The structure of TnC with two calcium ions bound has previously been solved by X-ray methods. Shown here is the solution structure of TnC which has been solved using 3D and 4D heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques. The 1H, 13C, and 15N backbone chemical shifts have already been published [Slupsky, C. M., Reinach, F. C., Smillie, L. B., & Sykes, B. D. (1995) Protein Sci. 4, 1279-1290]. Presented herein are the 1H, 13C, and 15N side-chain chemical shifts which are 80% complete. The structure of calcium-saturated TnC was determined on the basis of 2106 NOE-derived distance restraints, 121 phi dihedral angle restraints, and 76 psi dihedral angle restraints. The appearance of calcium-saturated TnC reveals a dumbbell-shaped molecule with two globular domains connected by a linker. The structures of the N-terminal and C-terminal domains are highly converged [backbone atomic root mean square deviations (rmsd) about the mean atomic coordinate position for residues 10-80 and 98-155 are 0.66 +/- 0.17 and 0.69 +/- 0.18 A, respectively]; however, the orientation of one domain with respect to the other is not well-defined, and thus each domain appears to be structurally independent. Comparison of the calcium-saturated form of TnC determined herein with the half-saturated form determined by X-ray methods reveals two major differences. First, there is a major structural change which occurs in the N-terminal domain resulting in the opening of a hydrophobic pocket presumably to present itself to its target protein troponin I. This structural change appears to involve only helices B and C which move away from helices N/A/D by the alteration of the backbone phi, psi angles of glutamic acid 41 from irregular in the crystal structure (-97 degrees, -7 degrees) to helical in the NMR calcium-saturated structure (-60 degrees, -34 degrees). The other difference between the two structures is the presence of a flexible linker between the two domains in the NMR structure. This flexible linker allows the two domains of TnC to adopt any orientation with respect to one another such that they can interact with a variety of targets.

[Conformational state of oncoprecipitin cyprein and its immunologic activity]

Bioorg Khim. 1995; 21: 905-11

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The effect of solution ionic strength, calcium ion concentration, and temperature on spatial structure of cyprein was examined by CD, UV, and fluorescence spectroscopy. The secondary structure of the cyprein molecule was calculated from CD spectra, and the prevalence of the beta-structure (85%) was shown. An irreversible conformational transition in the range 55-60 degrees C was found, which reduces the binding activity of cyprein in interaction with carcinoembryonic antigen (CEA) and anti-cyprein antibodies. In the latter case, the binding activity was reversibly restored. Cyprein was shown to be a calcium-binding protein. Binding of calcium by cyprein and increasing the ionic strength of solution affect only tertiary structure of the protein. At an ionic strength of solution close to physiological conditions, calcium-bound cyprein shows maximum binding to CEA and anti-cyprein antibodies. It was shown by difference UV spectroscopy that cyprein does not interact specifically with the monosaccharides of the carbohydrate chains of CEA: fucose, mannose, galactose, and N-acetylglucosamine.

[Study of the structure and mechanism of action of troponin C and calmodulin by "protein" engineering]

Biokhimiia. 1995; 60: 395-418

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The structure, properties and action mechanisms of troponin C and calmodulin are reviewed. The primary and tertiary structures of calcium-binding proteins and the mechanisms of Ca2+ binding are analyzed. The methods used for investigating the functional activity of Ca-binding proteins are compared. Molecular biology approaches for analyzing the role of various ligands in Ca2+ binding are described. The role of alpha-helices in the maintenance of the overall structure, Ca2+ binding and calmodulin and troponin C interaction with target proteins is discussed. Mutations directed at the change of the electric charge and hydrophobicity of calmodulin and troponin C are described and compared. Data on the incorporation of Cys residues into calmodulin and troponin C structure are presented. The use of Cys-containing mutants of Ca-binding proteins for the study of conformational changes and protein-protein interaction is analyzed.

The S-100 is a group of low molecular weight (10-12 kD) calcium-binding proteins highly conserved among vertebrates. It is present in different tissues as dimers of homologous or different subunits (alpha, beta). In the nervous system, the S-100 exists as a mixture composed of beta beta and alpha beta dimers with the monomer beta represented more often. Its intracellular localisation is mainly restricted to the glial cytoplasmic compartment with a small fraction bound to membranes. In this compartment the S-100 acts as a potent inhibitor of phosphorylation on several substrates including the synaptosomal C-Kinase and Tau, a microtubule-associated protein. The S-100 in particular conditions, after binding with specific membrane sites (Kd = 0.2 microM; Bmax = 4.5 nM), is able to modify the activity of adenylate cyclase, probably via G-proteins. In addition, the Ca2+ homeostasis is also modulated by S-100 via an increase of specific membrane conductance and/or Ca2+ release from intracellular stores. "In vitro" and "in vivo" experiments showed that lower (nM) concentrations of extracellular S-100 beta act on glial and neuronal cells as a growth-differentiating factor. On the other hand, higher concentrations of the protein induce apoptosis of some cells such as the sympathetic-like PC12 line. Finally, data obtained from physiological (development, ageing) or pathological (dementia associated with Down's syndrome, Alzheimer's disease) conditions showed that a relationship could be established between the S-100 levels and some aspects of the statii.

The three-dimensional structure of porcine pancreatic PLA2 (PLA2), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 phi torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1-62 and 72-124 is 0.75 +/- 0.09 A for the backbone atoms and 1.14 +/- 0.10 A for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA2 and PLA2 in the ternary complex is 3.5 A for the backbone atoms and 4.0 A for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal alpha-helix. Whereas free in solution Ala1, Leu2 and Trp3 are disordered, with the alpha-helical conformation with the alpha-amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA2. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA2 in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA2.

Reticulocalbin, an endoplasmic reticulum (ER)-resident Ca(2+)-binding protein, is a member of the EF-hand Ca(2+)-binding protein superfamily [Ozawa, M. and Muramatsu, T. (1993) J. Biol. Chem. 268, 699-705]. Reticulocalbin has six repeats of a domain containing the EF-hand motif. In addition, the protein has an amino-terminal leader sequence which serves as a signal for transfer of the protein into the lumen of the ER, and a His-Asp-Glu-Leu sequence at its carboxy terminus which functions as a signal to retain the protein in the ER. In this paper, we describe the genomic structure of this unique Ca(2+)-binding protein. Southern blot analysis of mouse genomic DNA revealed that there is a single copy of the reticulocalbin gene per haploid genome. The gene spans over 13 kilobase pairs and encodes six separate exons. Thus, reticulocalbin differs from the cytosolic Ca(2+)-binding protein calbindin D28 which also has six EF-hand motif domains, but the gene for which is divided into 11 exons. While there is some correlation between exon division and protein domain structure, these relationships are not as clear as they are in other genes. Comparison of the gene organization of reticulocalbin with that of other EF-hand proteins revealed that reticulocalbin diverged very early from other members of the EF-hand protein super-family.

The S100 family of calcium binding proteins contains approximately 16 members each of which exhibits a unique pattern of tissue/cell type specific expression. Although the distribution of these proteins is not restricted to the nervous system, the implication of several members of this family in nervous system development, function, and disease has sparked new interest in these proteins. We now know that the original two members of this family, S100A1 and S100B, can regulate a diverse group of cellular functions including cell-cell communication, cell growth, cell structure, energy metabolism, contraction and intracellular signal transduction. Although some members of the family may function extracellularly, most appear to function as intracellular calcium-modulated proteins and couple extracellular stimuli to cellular responses via interaction with other cellular proteins called target proteins. Interaction of these proteins with target proteins appear to involve cysteine residues (one in S100A1 and two in S100B), as well as a stretch of 13 amino acids, in the middle of the molecule called the linker region, which connects the two EF-hand calcium binding domains. In addition to the amino acid sequence and secondary structures of these proteins, the structures of the genes encoding these proteins are highly conserved. Studies on the expression of these proteins have demonstrated that a complex mixture of transcriptional and postranscriptional mechanisms regulate S100 expression. Further analysis of the function and expression of these proteins in both nervous and nonnervous tissues will provide important information regarding the role of altered S100 expression in nervous system development, function and disease.

Structures of the troponin C regulatory domains in the apo and calcium-saturated states.

Nat Struct Biol. 1995; 2: 784-9

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Regulation of contraction in skeletal muscle occurs through calcium binding to the protein troponin C. The solution structures of the regulatory domain of apo and calcium-loaded troponin C have been determined by multinuclear, multidimensional nuclear magnetic resonance techniques. The structural transition in the regulatory domain of troponin C on calcium binding involves an opening of the structure through large changes in interhelical angles. This leads to the increased exposure of an extensive hydrophobic patch, an event that triggers skeletal muscle contraction.

The three-dimensional structure of calmodulin in the absence of Ca2+ has been determined by three- and four-dimensional heteronuclear NMR experiments, including ROE, isotope-filtering combined with reverse labelling, and measurement of more than 700 three-bond J-couplings. In analogy with the Ca(2+)-ligated state of this protein, it consists of two small globular domains separated by a flexible linker, with no stable, direct contacts between the two domains. In the absence of Ca2+, the four helices in each of the two globular domains form a highly twisted bundle, capped by a short anti-parallel beta-sheet. This arrangement is qualitatively similar to that observed in the crystal structure of the Ca(2+)-free N-terminal domain of troponin C.

The solution structure of Ca(2+)-free calmodulin has been determined by NMR spectroscopy, and is compared to the previously reported structure of the Ca(2+)-saturated form. The removal of Ca2+ causes the interhelical angles of four EF-hand motifs to increase by 36 degrees-44 degrees. This leads to major changes in surface properties, including the closure of the deep hydrophobic cavity essential for target protein recognition. Concerted movements of helices A and D with respect to B and C, and of helices E and H with respect to F and G are likely responsible for the cooperative Ca(2+)-binding property observed between two adjacent EF-hand sites in the amino- and carboxy-terminal domains.

Sarcoplasmic calcium-binding proteins (SCPs) are members of the EF-hand calcium-binding protein family which are characterized by the presence of helix-loop-helix motifs in their amino acid sequence. SCPs have an M(r) of approximately 20,000, a pI of approximately 5 and interact with two to three calcium ions (Ca2+) with a KD of 10(-7) to 10(-8) M. Mg2+ ions antagonize Ca2+ ion binding in a complex manner so that these proteins are exquisitely fine-tuned to interfere with the Ca2+ signal. SCPs apparently fulfil no specific activatory function. They exhibit strong polymorphism, show a marked homology to coelenterate photoproteins (aequorin, luciferin) and have been found only in invertebrates, predominantly in muscle and neurons. In mollusks, SCPs are distributed in a tissue-specific manner, with immunoreactivity to SCP I-like isoforms localized in electrically silent neurons colocalized with serotonin, and immunoreactivity to SCP II-like isoforms exclusively present in muscle.

The role of glycine (residue 89) in the central helix of EF-hand protein troponin-C exposed following amino-terminal alpha-helix deletion.

Protein Sci. 1994; 3: 2089-96

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Because an N-terminal alpha-helical (N-helix) arm and a KGK-triplet (residues 88KGK90) in the central helix of troponin-C (TnC) are missing in calmodulin, several recent studies have attempted to elucidate the structure-function correlations of these units. Presently, with a family of genetically manipulated derivatives especially developed for this study and tested on permeabilized isolated single skeletal muscle fiber segments, we explored the specificities of the amino acid residues within the N-helix and the KGK-triplet in TnC. Noticeably, the amino acid compositions vary between the N-helices of the cardiac and skeletal TnC isoforms. On the other hand, the KGK-triplet is located similarly in both TnC isoforms. We previously indicated that deletion of the N-helix (mutant delta Nt) diminishes the tension obtained on activation with maximal calcium, but the contractile function is revived by the superimposed deletion of the 88KGK90-triplet (mutant delta Nt delta KGK; see Gulati J, Babu A, Su H, Zhang YF, 1993, J Biol Chem 268:11685-11690). Using this functional test, we find that replacement of Gly-89 with a Leu or an Ala could also overcome the contractile defect associated with N-helix deletion. On the other hand, replacement of the skeletal TnC N-helix with cardiac type N-helix was unable to restore contractile function. The findings indicate a destabilizing influence of Gly-89 residue in skeletal TnC and suggest that the N-terminal arm in normal TnC serves to moderate this effect. Moreover, specificity of the N-helix between cardiac and skeletal TnCs raises the possibility that resultant structural disparities are also important for the functional distinctions of the TnC isoforms.

On the optimization of the physicochemical distances between amino acids in the evolution of the genetic code.

J Theor Biol. 1994; 168: 43-51

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Using the simulated annealing technique we re-examine the role played by the minimization of the physicochemical distances between amino acids during the origin of the organization of the genetic code. The results are discussed in the context of the various hypotheses proposed to explain how amino acids were allocated in the genetic code.

The gene encoding the human calbindin-D9k has been cloned and the complete sequence established. The gene spans about 5.5 kilobases and is localized on the X-chromosome, consists of three exons and carries four Alu repeats. The promoter and 1300 base-pairs of 5' flanking region have been characterized. Besides a TATA box and two CAAT-like motifs a sequence related to a vitamin D response element was detected about 1.1 kilobases upstream from the promoter. A sequence positioned 50 nucleotides downstream from the promoter showed extensive homology to the estrogen response element at the same location within the rat calbindin-D9k gene. Two essential nucleotides within this region are changed when the rat and human sequences are compared. The human element failed to bind the estrogen receptor as determined by gel retardation assay. It is proposed that a two-nucleotide change within this region causes the gene to lack expression in human uterus and possibly placenta.

In the native calcium-binding protein calbindin D9k (M(r) 8.700; 75aa; 2 EF-hands), the backbone carbonyl oxygen of Glu60 coordinates the Ca2+ ion in the C-terminal site (site II). The carboxylate group of the same residue forms a hydrogen bond to a water molecule that constitutes a Ca2+ ligand in the N-terminal site (site I). The mutant E60D, with the charge-conserving substitution Glu60-->Asp, has been prepared to study the role of Glu60 in subjoining the two Ca(2+)-binding sites and its role in the cooperative Ca2+ binding. Ca(2+)-binding studies of the mutant show that the overall affinity for calcium has decreased by a factor of 38 in comparison with wild-type calbindin D9k. The largest reduction is seen in the first macroscopic binding step. The Ca2+ affinities for both sites in the protein are reduced to a similar extent. In contrast, the mutation leads to a large increase in the cooperativity of calcium binding. Differential scanning calorimetry has been used to determine the thermal stability which is almost as high as in the wild-type protein. Cadmium binding has been assessed with 1H and 113Cd NMR. X-ray crystallographic studies of the E60D mutant in its calcium-bound form show very small structural changes relative to the wild-type protein. Almost all differences are within the error limits of the method. The largest crystallographic effects are seen in the crystal packing. Two E60D molecules with slightly different structure are found in the asymmetric unit in contrast to the single molecule in the wild-type crystal.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the troponin complex. Implications of photocross-linking of troponin I to troponin C thiol mutants.

J Biol Chem. 1994; 269: 5725-9

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Ca2+ regulation of vertebrate-striated muscle contraction is initiated by conformational changes in the Ca(2+)-binding protein troponin C (TnC) and subsequent changes in the interaction of TnC with the inhibitory protein TnI. We have constructed mutants of rabbit skeletal muscle TnC in which natural Cys-98 was replaced by Leu, and a single Cys residue was introduced at position 12 (TnC12) or 89 (TnC89). Cys residues of mutant TnCs were derivatized with 4-maleimidobenzophenone and photocross-linked to TnI in binary TnC.TnI complexes. After digestion with CNBr or proteases, cross-linked peptides were purified and sequenced. TnC12 cross-linked at or near TnI Met-134 in a region known to be sensitive not only to occupancy of the regulatory Ca(2+)-binding sites of TnC but also to the contractile state of the thin filament. TnC89 cross-linked to TnI(108-113) in the inhibitory region. Taken together with earlier findings, these results indicate that in the TnC.TnI complex, both domains of TnC, as well as the linker region between them, make contact with the inhibitory region of TnI. Our data also indicate that the N- and C-terminal domains of TnC interact with opposite ends of the TnI inhibitory region.

Purification and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY.

Biochemistry. 1994; 33: 6732-8

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The S100 proteins CAPL and CACY are expressed in a tissue- and cell-specific manner and have been reported to be associated with the metastatic phenotype of tumor cells. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified large amounts of the recombinant human proteins in Escherichia coli. Several characteristics of native proteins are shown to correspond to those of the bacterially expressed proteins. Both are able to form homodimers in vitro, probably the biologically active species, but not heterodimers. The Ca(2+)-binding parameters were studied by flow offlysis at physiological ionic strength. Both isotherms show a maximum of two Ca2+ per protein and are insensitive to Mg2+, indicating that the sites are of the Ca(2+)-specific type. The isotherms show slight (CAPL, nH = 1.15) or pronounced (CACY, nH = 1.33) positive cooperativity with K0.5 values of 0.32 mM (CACY) and 0.15 mM (CAPL), indicating that the sites are of the low-affinity type. Conformational changes in the Tyr microenvironment of CACY indicate that Ca2+ binding induces a shift of Tyr to a less polar environment. Mg2+ does not affect the fluorescence properties nor does it induce a difference spectrum, thus suggesting that at physiological ionic conditions it does not interact with the protein. The Ca(2+)-induced difference spectra of CAPL are about 3 times smaller than those of CACY, suggesting that the additional Tyr84 in CACY is much more sensitive to Ca2+ than the two Tyr residues conserved in both proteins.

Depletion and recovery of the calcium-binding proteins calbindin and parvalbumin in the pigeon optic tectum following retinal lesions.

Brain Res. 1994; 661: 289-92

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Retinal lesions in pigeons produced a marked depletion of somata and neuropil staining for both calbindin-D28k and parvalbumin immunoreactivities in the contralateral optic tectum. Calbindin-like immunoreactivity reappeared in some tectal layers by 6 weeks postlesion, whereas paravalbumin-like immunoreactivity recovered almost completely after 5 weeks. These data indicate that the retinal input may control the expression of calbindin and parvalbumin in the pigeon optic tectum.

Analysis of calcium-dependent interaction between amino-terminal conserved region of calpastatin functional domain and calmodulin-like domain of mu-calpain large subunit.

J Biol Chem. 1994; 269: 18977-84

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Calpain requires Ca2+ both for proteolysis of its substrates and for interaction with its endogenous inhibitor, calpastatin. Although calmodulin-like domains (CaMLDs) of large and small subunits of calpain have been suggested to be the sites for Ca(2+)-dependent interaction with calpastatin, specificity and molecular basis of the interaction have remained unclear. We investigated the interaction between the CaMLD of human mu-calpain large subunit expressed in Escherichia coli and a 19-residue synthetic oligopeptide corresponding to the region A (the amino-terminal conserved acidic region) of one of the four repetitive functional domains of calpastatin. The recombinant CaMLD bound to the oligopeptide immobilized on Sepharose beads in a Ca(2+)-dependent manner. The CaMLD failed in binding to a mutant oligopeptide with one amino acid substitution. Enhancement of fluorescence intensity of a hydrophobic probe, 2-(p-toluidino)naphthalene-6-sulfonate, was observed upon incubating with the CaMLD and further increased by Ca2+. The Ca(2+)-dependent enhancement of fluorescence intensity was strongly suppressed by the wild type oligopeptide, but not by the mutant one. Kinetic experiments were performed with BIAcore where binding of the CaMLD to the oligopeptide immobilized on a biosensor chip was detected as real time signals of surface plasmon resonance. The determined dissociation constant (KD) was 3.1 x 10(-9) M. These results suggest that the region A of calpastatin binds to the CaMLD in a specific manner similar to interactions between calmodulin-binding peptides and calmodulin where hydrophobic properties are known to be important.

Psoriasin binds calcium and is upregulated by calcium to levels that resemble those observed in normal skin.

J Invest Dermatol. 1994; 103: 370-5

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Recently, we described a small molecular weight protein termed psoriasin that showed sequence similarity with the S100 calcium-binding proteins and that is highly upregulated in psoriatic epidermis as well as in primary human keratinocytes undergoing abnormal differentiation. Here we present evidence showing that natural and recombinant psoriasin binds calcium, as judged by the calcium overlay assay, and that it contains all the sequence features characteristic of the S100 family. Furthermore, [35S]-methionine labeling experiments showed that psoriasin synthesis is upregulated by 2 mM Ca++ (ratio Ca++/control at 88 h = 2.56) to levels that resemble those observed in unfractionated keratinocyte populations obtained from normal skin.

The effects of calcitriol and a novel calcitriol analogue, 22-oxacalcitriol (OCT) on duodenal Ca transport, calbindin-D9k mRNA, and calbindin-D9k content were studied in two animal models reflecting common human pathologies, namely arterial hypertension and chronic renal failure, as well as in normal rats. The hormone or its analogue were administered intraperitoneally to vitamin-D-replete rats. Active Ca transport was increased in both spontaneously hypertensive rats (SHR) and in normotensive control WKY rats 5 h after calcitriol dosing of either 60 and 600 ng per rat. In WKY, calbindin-D9k content was slightly increased after the injection of 60 ng calcitriol, but not of 600 ng calcitriol whereas calbindin-D9k mRNA stayed essentially unchanged. In contrast, active Ca transport was significantly stimulated after the higher dose of 600 ng calcitriol. In SHR, while both doses of calcitriol increased active Ca transport, they had no stimulatory effect on calbindin-D9k mRNA or protein. In chronically uraemic rats, active Ca transport, duodenal calbindin-D9k and calbindin-D9k mRNA were stimulated after the injection of two subsequent doses of 300 ng calcitriol per rat. OCT treatment at same dosage led to a similar stimulation of calbindin-D9k and calbindin-D9k mRNA, but failed to induce an increase in active Ca transport. These results show that the stimulation of intestinal active Ca transport and calbindin-D9k can be entirely dissociated at the protein synthesis and the mRNA expression level (1) after calcitriol administration to normal and hypertensive rats, and (2) after OCT administration to uraemic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The highly efficient production of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) in a bacterial expression system.

Arch Biochem Biophys. 1994; 308: 311-7

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We expressed gram amounts of full-length and mutant rat brain calcium-binding proteins (calbindins-D28K) lacking one or two "EF-hand" motifs in a bacterial expression system. The cDNA for the full-length rat calcium-binding protein was cloned into the NdeI and BamHI sites of the pET3a vector. Additionally, constructs of the rat brain calcium-binding protein lacking EF-hand 2 (delta 2 mutant), EF-hand 6 (delta 6 mutant), and EF-hands 2 and 6 (delta 2, 6 mutant) were constructed using the same vector. These chimeric plasmids were used to transfect BL21 (DE3) pLysS Escherichia coli cells. Following transformation, the cells were grown in the presence of isopropylthiogalactoside in order to induce bacterial T7 polymerase, which resulted in the production of large amounts of the proteins of interest in the bacterial cytosol. Expressed full-length and delta 2 and delta 2,6 mutant proteins represented 50% or more of total bacterial protein. The delta 6 protein was not expressed. Cell lysis followed by purification of the proteins on DEAE-cellulose routinely resulted in gram yields of the proteins. The purified proteins displayed the appropriate amino acid composition and amino-terminal amino acid sequence. When analyzed by matrix-assisted laser desorption mass spectrometry the proteins were found to have the appropriate molecular weights (within the accuracy limits of the instrument). The expressed proteins bound to a polyclonal antiserum raised against chick intestinal calcium-binding protein. In addition, the full-length, delta 2, and delta 2,6 mutants bound calcium as assessed by a 45Ca blotting procedure. The production of large amounts of readily purified vitamin D-dependent calcium-binding proteins should be useful in biophysical studies of the proteins.

Recoverin, a new member of the EF-hand superfamily, serves as a Ca2+ sensor in vision. A myristoyl or related N-acyl group is covalently attached at its N-terminus and plays an essential role in Ca(2+)-dependent membrane targeting by a novel calcium-myristoyl switch mechanism. The structure of unmyristoylated recoverin containing a single bound Ca2+ has recently been solved by X-ray crystallography [Flaherty, K. M., Zozulya, S., Stryer, L., & McKay, D. B. (1993) Cell 75, 709-716]. We report here multidimensional heteronuclear NMR studies on Ca(2+)-free, myristoylated recoverin (201 residues, 23 kDa). Complete polypeptide backbone 1H, 15N, and 13C resonance assignments and secondary structure are presented. We find 11 helical segments and two pairs of antiparallel beta-sheets, in accord with the four EF-hands seen in the crystal structure. The present NMR study also reveals some distinct structural features of the Ca(2+)-free myristoylated protein. The N-terminal helix of EF-2 is flexible in the myristoylated Ca(2+)-free protein, whereas it has a well-defined structure in the unmyristoylated Ca(2+)-bound form. This difference suggests that the binding of Ca2+ to EF-3 induces EF-2 to adopt a conformation favorable for the binding of a second Ca2+ to recoverin. Furthermore, the N-terminal helix (K5-E16) of myristoylated Ca(2+)-free recoverin is significantly longer than that seen in the unmyristoylated Ca(2+)-bound protein. We propose that this helix is stabilized by the attached myristoyl group and may play a role in sequestering the myristoyl group within the protein in the Ca(2+)-free state.

The effects of deletion of the amino-terminal helix on troponin C function and stability.

J Biol Chem. 1994; 269: 9857-63

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Troponin C has a 14-residue alpha-helix at the extreme amino terminus (the N-helix) which is absent in calmodulin. To learn the significance of this region in troponin C, residues 1-14 were deleted using site-directed mutagenesis. Analysis of the mutant troponin C (delta 14-TnC) showed that deletion of the N-helix did not alter the secondary structure of troponin C. Like wild type troponin C, it exhibited Ca(2+)-dependent conformational changes based on electrophoretic mobility and increases in alpha-helix content. The thermal stability of delta 14-TnC, however, was 20 degrees C lower than wild type troponin C in the presence or absence of divalent cations because of destabilization of the amino-terminal domain. To determine the functional consequences of the deletion, its ability to relieve troponin I and IT inhibition of the actomyosin ATPase was assayed. The results show that the mutant could relieve troponin I inhibition in the presence and absence of Ca2+ but could relieve troponin IT inhibition only to 45-50% of the wild type level, even at high concentrations. Also, the calcium affinity of the low affinity sites is reduced as evidence by the 2.4-2.8-fold increase in Ca2+ concentration required to achieve half-maximal activation of the MgATPase and calcium titration of the metal-induced conformation monitored by far UV circular dichroism measurements. In addition, the N-helix is required for the full conformational change to take place upon the binding of Ca2+, but not Mg2+, to the high affinity sites. The results indicate that the N-helix of troponin C is important for the stability of troponin C and may play a vital role in the Ca(2+)-switching mechanism.

Ca2+, Mg2+, and troponin I inhibitory peptide binding to a Phe-154 to Trp mutant of chicken skeletal muscle troponin C.

Biochemistry. 1994; 33: 2961-9

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The effects of Ca2+, Mg2+, and troponin I (TnI) inhibitory peptide (Ip) binding on the spectral properties of a Phe-154 to Trp mutant (F154W) of chicken recombinant troponin C (rTnC) have been examined. Residue 154 is positioned in the final flanking helix H of metal binding site IV. Since there are no other Tyr or Trp residues in the protein, spectral properties can be unambiguously assigned. No significant differences in the far UV CD spectra of rTnC and F154W were observed in either the absence or presence of Ca2+. When reconstituted into whole Tn the ATPase specific activities (+/- Ca2+) of the troponin-tropomyosin-actomyosin subfragment 1 system were the same for both proteins. A 2-fold reduction in Ca2+ affinity of C domain sites III/IV but not of N domain sites I/II in isolated F154W is explicable in terms of the environment of residue 154 in the relatively disordered apo-C domain and its buried position in the known ordered 2Ca2+ crystal structure. Filling of sites III/IV by divalent cations was accompanied by a number of spectral changes which were different for Ca2+ and Mg2+. Binding of Ip peptides (residues 96-116 and 104-115(116)) elicited fluorescence emission spectral alterations in the presence of Ca2+. These were not observed in its absence nor in the presence of Mg2+ even though binding occurs under these conditions. Since Ca2+ affinity to C domain but not to N domain sites was increased by Ip at the low concentrations of protein and Ip tested, Ip binding appears to be stronger with C domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Multidimensional heteronuclear NMR spectroscopy has been used to determine almost complete backbone and side-chain 1H, 15N, and 13C resonance assignments of calcium loaded Myxococcus xanthus protein S (173 residues). Of the range of constant-time triple resonance experiments recorded, HNCACB and CBCA(CO)NH, which correlate C alpha and C beta with backbone amide resonances of the same and the succeeding residue respectively, proved particularly useful in resolving assignment ambiguities created by the 4-fold internal homology of the protein S amino acid sequence. Extensive side-chain 1H and 13C assignments have been obtained by analysis of HCCH-TOCSY and 15N-edited TOCSY-HMQC spectra. A combination of NOE, backbone amide proton exchange, 3JNH alpha coupling constant, and chemical shift data has been used to show that each of the protein S repeat units consists of four beta-strands in a Greek key arrangement. Two of the Greek keys contain a regular alpha-helix between the third and fourth strands, resulting in an unusual and possibly unique variation on this common folding motif. Despite similarity between two nine-residue stretches in the first and third domains of protein S and one of the Ca(2+)-binding sequences in bovine brain calmodulin [Inouye, S., Franceschini, T., & Inouye, M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833], the protein S topology in these regions is incompatible with an EF-hand calmodulin-type Ca(2+)-binding site.

Solution structure of the TR1C fragment of skeletal muscle troponin-C.

J Biol Chem. 1994; 269: 6773-8

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Residues 12-87 (TR1C fragment) of turkey skeletal muscle troponin-C comprises two helix-loop-helix calcium-binding motifs which are the regulatory calcium-binding sites in the N-terminal domain of the protein. We have used the combined distance geometry-simulated annealing protocol DGII (Havel, T. F. (1991) Prog. Biophys. Mol. Biol. 56, 43-78) to determine the structure of this 76-residue polypeptide in solution from 475 1H NMR-derived distance restraints. The nuclear Overhauser enhancement-derived distance constraints used in the DGII protocol were supplemented by introducing generic hydrogen bond distance restraints for slowly exchanging amide hydrogens in regular secondary structure elements, by restricting the available phi angle space to -180 degrees to 0 degrees for all residues except glycines, and by tailoring the distance boundaries used for quantitating the nuclear Overhauser enhancement intensities to correspond to characteristic distances found in helices. This improved the geometry of the four helices in the resulting structures. The relative positions of helices A and B which flank calcium-binding loop 1, helix D which follows calcium-binding loop 2, and the beta-sheet between the two calcium-binding loops were well defined and had an overall root-mean-square deviation for 20 converged structures of 1.4 +/- 0.2 A for backbone atoms. The structure and relative orientations of these regions are very similar to these of the corresponding regions of the protein in the crystal structure of intact turkey skeletal troponin C (Herzberg, O., and James, M. N. G. (1988) Nature 313, 653-659). The structure of helix C was well defined, but its relative position to the other helices was not defined. It occupied a range of positions in the set of 20 DGII structures, the average of which was quite similar to the orientation of helix C in the x-ray structure. The overall structure of the apo regulatory domain of troponin-C is therefore not affected by the loss of the N-helix, or the low pH conditions used for the x-ray structure, but may be more flexible in regions known to be involved in contacts with other skeletal muscle regulatory proteins.

The two globular N and C domains of chicken troponin C (TnC) are connected by an exposed alpha-helix (designated D/E; residues 86-94). Recombinant N (residues 1-90) and C (residues 88-162) domains containing either F29 or W29 and F105 or W105 have been engineered and expressed in Escherichia coli. These termination and initiation sites were chosen to minimize disruption of side-chain interactions between the D/E helix and other residues. W29 and W105 served as useful spectral probes for monitoring Ca(2+)-induced structural transitions of the N and C domains, respectively [Pearlstone et al. (1992) Biochemistry 31, 6545-6553; Trigo-Gonzalez et al. (1992) Biochemistry 31, 7009-7015]. By all criteria tested, the properties of the isolated F29W/N domain (1-90) were identical to those of the N domain in intact F29W. These included fluorescence emission spectra in the absence and presence of Ca2+/Mg2+, far-UV CD spectra, and Ca2+ affinity as monitored by fluorescence and ellipticity at 221 nm. Similar but not identical properties were observed for isolated F105W/C domain (88-162) and intact F105W. A summation of the far-UV CD spectra (+/- Ca2+) of the two domains was virtually superimposable on that of the intact protein. Of the total Ca(2+)-induced ellipticity change at 221 nm, 27% could be assigned to the N domain and 73% to the C domain. The data suggest a significant Ca(2+)-induced transition involving secondary structural elements of the N domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a novel calcium-binding protein, S100C, from porcine heart.

Biochim Biophys Acta. 1994; 1223: 348-53

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A novel Ca(2+)-binding protein, which we have named S100C (Ohta et al. (1991) FEBS Lett. 295, 93-96), was purified to homogeneity from porcine heart by Ca(2+)-dependent dye-affinity chromatography. S100C possesses some properties of S100 proteins, such as self-association and exposure of a hydrophobic site upon binding of Ca2+ but it differs from S100 proteins in forms of its isoelectric point (pI = 6.2), cross-reactivity with antibodies, staining by Stains-all, and its Ca(2+)-dependent interaction with the immobilized dye. S100C bound to cytoskeletal components at physiological concentrations of Ca2+. Moreover, it was found that 125I-labeled S100C interacted with annexin I in a Ca(2+)-dependent manner. S100C also inhibited the phosphorylation of annexin I by protein kinase C. These data suggest that S100C might act to regulate the cytoskeleton in a Ca(2+)-dependent manner via interactions with annexin I.

A three-dimensional solution structure of amicyanin from Thiobacillus versutus has been determined by distance geometry and restrained molecular dynamics. A total of 984 experimentally derived constraints were used for the final refinement (881 distance constraints and 103 dihedral angle constraints). Stereospecific assignments were made for 17 prochiral beta-methylene protons (33%) and the methyl groups of eight valine residues. Fourteen structures were selected to represent the solution structure. They show an average pairwise backbone root-mean-square deviation of 1.19 A. The overall structure can be described as a beta-sandwich, built up of nine beta-strands. The copper atom is located between three loops on one end of the molecule. Two of these loops contribute the copper ligands. His54 is on the loop between beta-strands 4 and 5. The other three ligands, Cys93, His96 and Met99, are located evenly spaced on the loop between beta-strands 8 and 9. This loop is folded in two consecutive type 1 turns with His96 as the donor and acceptor of the NHi-CO(i-3) hydrogen bonds. The folding is reminiscent of the general cupredoxin fold. Considerably different are the large 21 residue N-terminal extension, that is unique to amicyanin and forms an extra beta-strand (strand 1), and the region between beta-strands 5 and 7. The partly surface-exposed copper ligand His96 is surrounded by a hydrophobic patch consisting of seven residues.

The crystal structure of recoverin reveals two functional Ca2+-binding sites and a surface hydrophobic feature that may be important for membrane-binding and recoverin's role in the rod cell's recovery from light.

Troponin and its Ca(2+)-binding subunit troponin C (TnC) of the Ezo-giant scallop, Patinopecten yessoensis, have been revealed to bind only 1 mol of Ca2+/mol irrespective of the presence and absence of Mg2+. The amino acid sequence of the TnC has been determined by the automated Edman degradation. TnC is composed of 152 residues including 3 tryptophans at positions 53, 70, and 109, 4 cysteines at positions 19, 31, 67, and 145, and no proline. The molecular weight is calculated as 17,410. The NH2 terminus of TnC is blocked by an acetyl group. The sequence of scallop TnC required deletion of three residues in the D/E linker region to maximize sequence homology to other TnCs and shows considerably lower homology to vertebrate skeletal TnCs (27-30%), ascidian TnC (26%), arthropoda TnCs (30-37%), and chicken calmodulin (37%). Further, we conclude that Ezo-giant scallop TnC binds Ca2+ at site IV, a site specific for Ca2+. The other sites I, II, and III appeared to lose the Ca2+ binding ability due to substitutions of some important residues.

Binding of Ca2+ to calbindin D9k: structural stability and function at high salt concentration.

Biochemistry. 1994; 33: 14170-6

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Calcium binding constants of wild-type calbindin D9k and mutant forms with one, two, and three neutralized negative charges in the vicinity of the Ca2+ binding sites are determined at varying KCl concentrations from 2 mM to 1 M. The results indicate that the added salt does not cause significant structural changes in calbindin D9k and, along with site-directed mutagenesis, can be used as a well-controlled means for modulating electrostatic interactions. The lack of structural changes at high salt concentrations is also supported by two-dimensional 1H NMR data. High salt concentrations are observed to substantially reduce the cooperativity of calcium binding to calbindin D9k. This suggests that the cooperativity is strongly dependent on electrostatic interactions. The data have been used to test a dielectric continuum model for protein electrostatics using a macroscopic dielectric constant of water throughout the system. Excellent agreement between experiment and Monte Carlo simulations is observed for the whole set of data covering changes in the binding constant of more than 6 orders of magnitude. A simplified theoretical treatment using the Kirkwood-Tanford formula, based on the Debye-Huckel approximation, yields an almost equally good agreement with the experiment.

Disturbed calcium homeostasis may play a role in the etiology in Alzheimer's and other neurodegenerative diseases. A protective role against cellular degeneration has been postulated for Ca(2+)-binding proteins in certain neuron populations. Recent data suggest that intracellular free calcium regulation is also altered in several non-neuronal cells, including skin fibroblasts, from patients with Alzheimer's disease. In this study we analyzed the expression of several EF-hand Ca(2+)-binding proteins in cultured skin fibroblasts from Alzheimer patients and age-matched normal donors. We detected a strong expression of some members of the S100 Ca(2+)-binding protein family and of calcineurin A. However, no significant differences were found between both types of donors by Northern blot and Western blot analysis. In addition, similar signals were detected on 45Ca(2+)-blots of fibroblasts extracts of Alzheimer patients and control donors. The present findings indicate that the altered level of some intracellular calcium-binding proteins in certain brain areas of Alzheimer patients is not found in skin fibroblasts of these patients.

Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins.

J Biol Chem. 1994; 269: 32807-13

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VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of a calbindin-D9K-like protein in avian muscle cells by 1,25-dihydroxy-vitamin D3.

Biochem Mol Biol Int. 1994; 32: 859-67

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1,25-dihydroxy-vitamin D3 induces the synthesis of a 9 kDa calcium binding protein in chick embryo myoblasts. This work revealed comigration of the myoblast protein with rat calbindin-D9K in SDS-polyacrylamide gels. Western-blot analysis with a specific calbindin-D9K antibody showed the presence of an immunoreactive protein of 9 kDa in chick tissues. In addition, higher levels of myoblast mRNA hybridizing to a specific cDNA probe for rat calbindin-D9K were detected in response to 1,25-dihydroxy-vitamin D3. Northern hybridization analysis showed that the increase was related to a single 550 nucleotide mRNA species. These results provide the first evidence on the presence of calbindin-D9K or a closely related protein in avian tissues and its inducible expression in muscle by 1,25-dihydroxy-vitamin D3.

The endoplasmic reticulum calcium-binding protein of 55 kDa is a novel EF-hand protein retained in the endoplasmic reticulum by a carboxyl-terminal His-Asp-Glu-Leu motif.

J Biol Chem. 1994; 269: 19142-50

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We have identified a new human Ca(2+)-binding protein that is specifically localized in the endoplasmic reticulum (ER). The protein is termed ERC-55, i.e. ER calcium-binding protein of 55 kDa. ERC-55 is a single copy gene and is encoded by an approximately 1900-base mRNA, which shows a ubiquitous expression pattern. The ERC-55 protein comprises an amino-terminal signal sequence followed by six copies of the EF-hand Ca2+ binding motif. Ca2+ binding was demonstrated directly for recombinant ERC-55 using the 45Ca2+ overlay technique. The carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL) is required for retention of ERC-55 in the ER. Deletion of HDEL results in slow secretion into the medium. In pulse-chase experiments, approximately 50% of the HDEL deletion mutant is secreted, whereas no detectable secretion is observed with the wild-type protein. This represents the first example of an endogenous human protein that is retained in the ER by an HDEL rather than Lys-Asp-Glu-Leu (KDEL) carboxyl-terminal tetrapeptide. Comparative sequence analysis indicates that ERC-55, together with the recently identified protein reticulocalbin (Ozawa and Muramatsu, 1993), constitute a new subfamily of the EF-hand superfamily of Ca(2+)-binding proteins that are specifically located in the ER.

Differential feeding-related regulation of ubiquitin and calbindin9kDa in rat duodenum.

Biochim Biophys Acta. 1994; 1200: 191-6

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Analyses of the calcium-binding protein, calbindin9kDa, purified to apparent homogeneity (SDS-PAGE) from rat duodenum, revealed variable contamination by two other 9 kDa proteins (up to 0.2 mol equivalent each) which were identified as ubiquitin and its C-terminal variant, des-Gly-Gly-ubiquitin. We found that the co-purification of these proteins did not reflect a tight molecular interaction but instead their unexpectedly similar physical characteristics in nondenaturing conditions. Like calbindin9kDa, free ubiquitin was abundant (1% and 0.4% of soluble protein, respectively) in duodenum mucosa of 7-8-week-old rats and its concentration varied daily and with feeding status. In rats fed from midnight to 8.30 a.m., the ubiquitin concentration was specifically higher at 10 pm than at 10 a.m. (11.2 +/- 0.7 and 7.7 +/- 0.8 nmol per g wet weight, respectively, P < 0.02), whereas calbindin9kDa tended towards an opposite variation (18.0 +/- 1.9 and 21.8 +/- 1.7 nmol per g, respectively). Based on its unusually high abundance and novel feeding-related variations, ubiquitin must have an important functional role in the rat duodenum which is distinctly regulated from the calcium transport-associated role of calbindin9kDa.

Characterization of the lanthanide ion-binding properties of calcineurin-B using laser-induced luminescence spectroscopy.

Biochemistry. 1994; 33: 10428-36

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Calcineurin (CaN) is a Ca2+/calmodulin-dependent protein phosphatase found in brain and other tissues. It is a heterodimer consisting of a catalytic subunit (CaN-A) and a Ca(2+)-binding regulatory subunit (CaN-B). The primary structure of CaN-B indicates that it, like calmodulin, is an EF-hand protein and binds four Ca2+ ions. Eu3+, due to its favorable spectroscopic and chemical properties, has been substituted for Ca2+ in CaN-B to determine the metal ion-binding properties of this "calmodulin-like" protein. Excitation of the 7F0-->5D0 transition of Eu3+ results in a spectrum similar to that of calmodulin, consisting of three peaks. Analysis of the spectral titration curves reveals four Eu(3+)-binding sites in CaN-B. The affinities vary: sites I and II have dissociation constants of 1.0 +/- 0.2 and 1.6 +/- 0.4 microM, respectively; the values for sites III and IV are Kd = 140 +/- 20 and Kd = 20 +/- 10 nM, respectively. Binding of Tb3+ is slightly weaker. Tb3+ luminescence, sensitized by tyrosine, reveals that for lanthanides the highest affinity sites lie in the C-terminal domain. Energy transfer distance measurements between Eu3+ and Nd3+ in sites III and IV reveal a separation of 10.5 +/- 0.5 A, which suggests that these sites are arranged in a typical EF-hand pair. This information indicates that the overall structure of CaN-B is similar to the dumbbell-shaped proteins troponin-C and calmodulin, but is more like TnC in its metal-binding properties.

Intrinsic tyrosines, as monitored by fluorescence spectroscopy, are sensitive reporters of local, Ca(2+)-induced conformational changes in troponin C (TnC). Rabbit skeletal TnC contains two tyrosines (Y10 in the N-helix, and Y109 in site 3 in the C-terminal domain) in distinct microenvironments: their individual contributions to total fluorescence intensity are elucidated here utilizing bacterially synthesized rabbit skeletal TnC (sTnC4) and a genetically engineered variant, termed 109YF, lacking one of the tyrosines (Y109 replaced with F109). The steady-state fluorescence emission spectra following excitation at 280 nm were recorded in EGTA (Ca(2+)-free) and Ca(2+)-saturated (pCa4) solutions. For the wild-type sTnC4, pCa4 causes a significant (46%) increase in the peak fluorescence intensity over the value in EGTA. For the mutant 109YF, the EGTA fluorescence is only marginally affected (74% of the wild-type FEGTA), but interestingly the Ca2+ effect is completely suppressed (delta F = FpCa4-FEGTA = 2% of the wild-type value). These results indicate that the two tyrosines make disparate contributions to the fluorescence spectrum of wild-type sTnC, both in the presence and absence of Ca2+; whereas Y10 in the N-helix is dominant in Ca(2+)-free solution, Y109 is the sole contributor to the Ca2+ effect. Furthermore, to explain the biphasic fluorescence response of Y109 obtained during Ca2+ titrations, the findings yield the most unequivocal evidence that Ca(2+)-induced conformational changes in the trigger sites operating the contractile switch modify properties of the C-terminal sites in TnC pari passu.

The 10 Met methyl groups in recombinant cardiac troponin (cTnC) were metabolically labeled with [13C-methyl]Met and detected as 10 individual cross-peaks using two-dimensional heteronuclear single- and multiple-quantum coherence (HSMQC) spectroscopy. The epsilon C and epsilon H chemical shifts for all 10 Met residues were sequence-specifically assigned using a combination of HSMQC and systematic conversion of the Met residues to Leu. The only negative functional consequence of these changes was seen when both Met 45 and 81 were mutated. Binding of Ca2+ to the high affinity C-terminal sites III and IV induced relatively large changes in the epsilon H and epsilon C chemical shifts of all Met residues in the C-terminal domain as well as small but significant changes in the chemical shifts of epsilon H Met 47 and Met 81 in the N-terminal half of cTnC. Binding of Ca2+ to the low affinity N-terminal site II induced large changes in the epsilon H and epsilon C chemical shifts of Met 45, Met 80, and Met 81. Binding of Ca2+ to site II had no effect on the chemical shifts of Met residues located in the C-terminal domain. The nature of the chemical shift changes of Met residues in the N- versus the C-terminal halves of cTnC were consistent with different Ca(2+)-induced conformational changes in these domains. Thus, the assigned methyl Met chemical shifts can serve as useful structural markers to study conformational transitional in free cTnC and potentially after association with small ligands, peptides, and other troponin subunits.

Reductive methylation and pKa determination of the lysine side chains in calbindin D9k.

J Protein Chem. 1994; 13: 527-35

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The Lys residues in the 75-residue Ca(2+)-binding protein calbindin D9k were reductively methylated with 13C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca(2+)- and apo-forms of the protein, the 13C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. The pKa values of the individual Lys residues in Ca(2+)-calbindin D9k and apo-calbindin D9k were obtained by combining pH titration experiments and (1H, 13C)-HMQC NMR spectroscopy. Each Lys residue in the Ca(2+)- and apo-forms of calbindin D9k has a unique pKa value. The Lys pKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D9k has a very similar structure compared to Ca(2+)-calbindin D9k, the removal of two Ca2+ ions from the protein leads to an increase of the pKa values of the Lys residues.

Molecular cloning of a novel calcium-binding protein structurally related to hippocalcin from human brain and chromosomal mapping of its gene.

Biochim Biophys Acta. 1994; 1222: 515-8

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A cDNA clone (hHLP2) encoding a novel calcium-binding protein structurally related to hippocalcin has been isolated from the human hippocampus cDNA library. The primary structure consists of 193 amino acids, and contains three EF-hand structures and a possible NH2-terminal myristoylation site. A single transcript at a position corresponding to 1.7 kilobases was detected only in the brain. The hHLP2 gene was mapped to human chromosome 2.

Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I.

J Biol Chem. 1994; 269: 5230-40

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Calcium binding to regulatory sites located in the NH2-terminal domain of troponin C (TnC) induces a conformational change that blocks the inhibitory action of troponin I (TnI) and triggers muscle contraction. We used deletion mutants of TnI in conjunction with a series of TnC mutants to understand the structural and functional relationship between different TnI regions and TnC domains. Our results indicate that TnI is organized into structural and regulatory regions which interact in an antiparallel fashion with the corresponding structural and regulatory regions of TnC. Functional studies show that the COOH-terminal region of TnI, when linked to the inhibitory region (TnI103-182) can regulate actomyosin ATPase. A TnI lacking the first 57 amino acids (TnId57) has been shown to have similar properties (Sheng, Z., Pan, B.-S., Miller, T. E., and Potter, J. D. (1992) J. Biol. Chem. 267, 25407-25413). Regulation was not observed with the COOH-terminal region alone (TnI120-182), with the NH2-terminal region alone (TnI1-98), or with the NH2-terminal linked to the inhibitory region (TnI1-116). Binding studies show that the NH2-terminal region of TnI interacts with the COOH-terminal domain of TnC in the presence of Ca2+ or Mg2+ and that the inhibitory plus COOH-terminal region of TnI (TnI103-182) interacts with the NH2-terminal domain of TnC in a Ca(2+)-dependent manner. Based on these results we propose a model for the Ca(2+)-induced conformational change. In our model the NH2-terminal domain of TnI is anchored strongly to the COOH-terminal domain of TnC in the absence and presence of Ca2+ while the inhibitory and COOH-terminal regions of TnI switch between actin-tropomyosin in the absence of Ca2+ to binding sites in both NH2- and COOH-terminal domains of TnC in the presence of Ca2+.

The three-dimensional NMR solution structure of alpha-cobratoxin at pH 7.5 and conformational differences with the NMR solution structure at pH 3.2.

J Biomol Struct Dyn. 1994; 12: 1-17

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The 3D solution structure of alpha-cobratoxin, a neurotoxin purified from the Naja naja siamensis snake venom, has been determined by Nuclear Magnetic Resonance spectroscopy, in conjunction with distance geometry and restrained molecular dynamics, at pH 7.5. A total of 490 distance restraints were obtained from NOE intensities and 25 phi dihedral angle restraints deduced from J-coupling data. The generated structures are well defined with root mean square deviations from a geometrical mean structure of 0.107 +/- 0.036 nm for the backbone atoms and 0.128 +/- 0.073 nm for the side-chain atoms (considering residues 1 to 66 minus 26 to 35). A comparison between the generated structures at pH 7.5 and the mean NMR solution structure at pH 3.2 revealed that the 3D structure of alpha-cobratoxin is more compact at neutral pH. This major difference is mainly due to the pH-dependent conformational variations of three residues His18, Thr44 and Thr59.

High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin.

J Biol Chem. 1994; 269: 15795-802

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Caltractin is a member of the calmodulin superfamily of Ca(2+)-binding proteins that was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii. Human and mouse homologs to algal caltractin have been recently characterized. In the studies reported here, recombinant Chlamydomonas caltractin was expressed at high levels in Escherichia coli and purified to homogeneity. The use of the ompT-host BL21 proved critical for obtaining high yields of homogeneous full-length protein. Growth and purification protocols were optimized to allow reproducible and efficient production of tens of milligrams of pure protein from 1-liter cultures. Caltractin has a distinct UV spectrum which is largely dominated by the fine structure due to the 9 Phe residues. Unlike other members of the same protein family, the UV and the CD spectra do not change upon addition of Ca2+ to the apoprotein. However, the 1H NMR spectrum shows distinct changes upon Ca2+ binding, which are indicative of structural and/or dynamic changes largely reminiscent of other members of the calmodulin superfamily. Ca2+ binding measurements demonstrated the binding of four Ca2+ ions to caltractin with two higher affinity (Kd = 1.2 x 10(-6) M) and two lower affinity (Kd = 1.6 x 10(-4) M) sites. Caltractin is highly stable in both the apo- and the Ca(2+)-loaded states. The unusual stability of apocaltractin makes this protein highly suited for structural studies by multidimensional NMR aimed at understanding the structural and dynamic consequences of Ca2+ binding, and the molecular basis of Ca2+ signal transduction.

The solution structure of the 56 amino acid residue turkey ovomucoid third domain was determined by n.m.r. methods. Of the 661 distance constraints used in the calculations, 120 were determined by quadratic approximation of the cross-relaxation rates. The remaining constraints were crudely estimated from a more standard analysis of NOESY spectra. Additionally, 29 torsion angle constraints, 17 hydrogen bonds, and three disulfide bridges were used in the structure calculations. Stereospecific assignments were accomplished for 24 beta-methylene groups and six isopropyl methyl groups (43% chiral assignments). The addition of more accurate distance constraints to the distance geometry/simulated annealing approach resulted in a significant reduction in the dispersion of calculated backbone torsion angles and root-mean-square deviations between structures. Detailed comparisons have been made between the n.m.r. structures of OMTKY3 and published X-ray structures of the same protein and of closely related avian ovomucoid third domains. The refinement with more accurate distance constraints reduced differences between families of the n.m.r. and the X-ray structures.

A functional and degenerate pair of EF hands contains the very high affinity calcium-binding site of calbindin-D28K.

J Biol Chem. 1993; 268: 20917-22

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Calbindin-D28K, a member of the troponin C superfamily of calcium-binding proteins, had six putative EF hand domains containing one very high affinity and two to three lower affinity calcium-binding sites. The location and binding activity of the calcium-binding sites were examined with a recombinant calbindin-D28K protein. This protein (Calb I-II) only contained EF hand domains 1 and 2 of calbindin-D28K. Binding of calcium and calcium analogs, the lanthanides, by the recombinant protein was determined in fluorescence emission experiments. Calb I-II bound 1 mol of terbium/mol of protein. Terbium was displaced from Calb I-II by other lanthanides and calcium. Fluorescence from terbium was not quenched by holmium. These results and Hill plots of calcium binding activity, determined from intrinsic protein fluorescence measurements, indicated the presence of a single high affinity calcium-binding site on Calb I-II. The properties of the binding site indicated that the very high affinity site of calbindin-D28K was located in EF hand domains 1 and 2 of the protein. In addition, these findings indicated the NH2-terminal pair of EF hands in calbindin-D28K did not depend on interactions with other domains in the protein for high affinity calcium binding activity. The results suggested at least one calcium-binding domain of calbindin-D28K can exist as an independent EF hand pair.

A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.

Anal Biochem. 1993; 210: 1-6

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In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent attachment of chromophores to the loop that could subsequently be tested for their ability to sensitize the luminescence of Tb3+ or Eu3+ bound in the loop. 7-Diethylamino-3-((4'-iodoacetylamino)phenyl)-4-methylcoumarin was the most efficient Eu3+ sensitizer studied, consistent with a mechanism of energy transfer that involves the triplet state of the donor. 4-Iodoacetamidosalicylic acid was the most efficient Tb3+ donor tested. Levels of lanthanide ion and labeled C3 as low as 5 x 10(-10) mol/liter could be detected. This protein chelator system has potential to be a useful, flexible complement to the organic chelators currently used in lanthanide-based, time-resolved luminescence immunoassays.

The Ca(2+)-binding parameters of recombinant human calmodulin-like protein (CLP), a protein specifically expressed in mammary epithelial cells, were studied by flow dialysis in the absence and presence of 2, 10, and 30 mM MgCl2. In general, the four intrinsic binding constants (K'Ca) are about 8-fold lower than in animal and plant calmodulins. In the absence of Mg2+ the K'Ca values of the four binding steps equal 4.0 x 10(3), 3.3 x 10(4), 1.0 x 10(4), and 6.0 x 10(3) M-1, respectively. They allow us to distinguish two pairs of sites: a higher affinity pair with strong positive cooperativity and a lower affinity pair composed of non-interacting sites with different affinities. Mg2+ antagonizes Ca2+ binding by decreasing only Ca(2+)-binding steps 2 and 3, so that at high Mg2+ concentrations the positive cooperativity in the high-affinity pair has been lost and that the four K'Ca values are very similar with a mean K'Ca of 4 x 10(3) M-1. Direct Mg2+ binding studies by equilibrium gel filtration indicate that 4-5 Mg2+ bind to CLP with a mean K'Mg of 250 M-1. Conformational changes in the unique Tyr138 microenvironment, monitored by fluorimetry and near-UV difference spectrophotometry, indicate that in metal-free CLP this Tyr is shielded from the polar solvent and strongly quenched by a specific chemical group; Ca2+ binding induces a shift of Tyr to a more polar environment and removal of the quenching group, but without full exposure to the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium ion is thought to play an important role in the intracellular signal transduction system and transfer its information through Ca(2+)-receptive proteins. Among many calcium receptive proteins, EF-hand type calcium receptive protein family are being investigated quite extensively. Calmodulin, one of representative EF-hand protein, localizes ubiquitously in many cells and tissues. This protein is involved in a general signal transduction system, such as, cell proliferation and differentiation. On the other hand, S-100 protein are distributed in specific cells and play specified roles.

Calcium ions regulate many processes in the central nervous system via interaction with intracellular calcium-binding proteins. One class of these proteins shares a common structural motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of many new members of this family. Some of these proteins, like parvalbumin, calbindin, and calretinin, proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major role is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. Cellular degeneration is often accompanied by Ca2+ overload. It has been assumed that neurons containing certain intracellular Ca(2+)-binding proteins may have a greater capacity to buffer Ca2+ and therefore would be more resistant to degeneration.

A refinement of the oncomodulin crystal structure at 1.30 A resolution has been carried out with X-ray data from the recombinant protein. The crystallographic R-factor values are 0.169 for 19,995 reflections in the range 6.0 to 1.30 A, which were used for the restrained least-squares refinement, and 0.176 for 20,186 observed reflections in the range 10.0 to 1.30 A. This high resolution refinement has enabled us to make more definitive statements about the molecular structure than was possible heretofore. The present model includes residues 1 to 108, the two Ca2+ of the CD and EF loops, two intermolecular Ca2+, and 103 water molecules per oncomodulin molecule. The electron density maps indicate disordered orientations for ten residues on the hydrophilic surface of the molecule. The pattern of molecular aggregation via intermolecular Ca2+, which occurs in the native rat oncomodulin structure, is also present in the recombinant oncomodulin structure. The Cys18 side-chain is not in a position that would be easily accessible for molecular dimerization via a disulphide bond. The substitution of Glu59, which is preserved in all the determined species of parvalbumin, by Asp59 in oncomodulin seems to break a stabilizing hydrogen bond in the CD loop and render the main-chain in positions 59 to 60 somewhat unstable. This instability in the CD loop, and the strong tendency of oncomodulin for molecular aggregation via intermolecular Ca2+, appear to be the two outstanding features that may account for oncomodulin's biological peculiarities.

We have purified recombinant human calretinin (CR) from Escherichia coli lysates and have produced a polyclonal antiserum against it. The antiserum recognizes determinants conserved in fish, chicken, rat, monkey and human CR. We show its use in the qualitative detection of CR by different methods of immunohistochemistry as well as in the detection of CR on immunoblots.

An invertebrate calcium-binding protein of the calbindin subfamily: protein structure, genomic organization, and expression pattern of the calbindin-32 gene of Drosophila.

J Neurosci. 1993; 13: 2186-98

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Antisera against vertebrate calcium-binding proteins cross-react with Drosophila nervous and muscle tissue. We have used an antiserum against carp parvalbumin to isolate from a Drosophila head cDNA library immunopositive expression clones. Tissue in situ hybridization identified a clone that labeled specific neurons and muscles similar to the parvalbumin-like immunohistochemical staining pattern. Five independent cDNAs derive from an mRNA whose open reading frame codes for a 310 amino acid polypeptide. Sequence analysis identifies six EF-hand calcium-binding domains and reveals 42% and 37% homology to chicken calretinin and calbindin D-28k, respectively. Since the positions of 9 out of 10 introns within the ORF are conserved from the Drosophila gene to both vertebrate genes, we conclude that we have identified the first invertebrate member of the calbindin sub-family of calcium-binding protein genes of the EF-hand homolog family. The calbindin-32 gene (cbn) maps to 53E on the second chromosome. It is expressed through most of ontogenesis with a selective distribution in the nervous system and in a few small adult thoracic muscles. The cloning of a Drosophila homolog to vertebrate neuronal Ca(2+)-binding proteins opens new routes to study the so far largely elusive function of these brain molecules.

A novel Ca(2+)-binding protein, tentatively designated reticulocalbin, has been identified and characterized. Reticulocalbin is a luminal protein of the endoplasmic reticulum with an M(r) of 44,000 as revealed by biochemical analysis and immunofluorescence staining. The cDNA of reticulocalbin encodes a protein of 325 amino acids with an amino-terminal signal sequence of 20 amino acids. The protein has six repeats of a domain containing the high affinity Ca(2+)-binding motif, the EF-hand. Although oxygen-containing amino acids important for the positioning of Ca2+ are conserved in all six domains, the conserved glycine residues in the central portion of the EF-hand motif are absent in three of them. Calcium blots showed that recombinant reticulocalbin expressed in bacterial cells binds Ca2+. The protein has the sequence His-Asp-Glu-Leu (HDEL) at its carboxyl terminus. This is similar to the Lys-Asp-Glu-Leu sequence, which serves as a signal to retain the resident proteins in the endoplasmic reticulum of animal cells. A mutant protein lacking the HDEL sequence produced by in vitro mutagenesis has been shown to be secreted into medium in transient expression assays.

Binding of two Ca2+ to the regulatory sites I and II of troponin C (TnC) induces a conformational transition believed to be responsible for the activation of muscle contraction. Based on the known crystal structure (2Ca2+ state), a model for the transition to the 4Ca2+ state has been proposed [Herzberg, O., Moult, J. & James, M. N. G. (1986) J. Biol. Chem. 261, 2638-2644]. The proposed conformational transition predicts that during Ca2+ binding a number of nonpolar residues become exposed to the solvent, creating a hydrophobic patch. Such a model implies that mutation of the hydrophobic to polar residues should increase the Ca2+ affinity at the regulatory sites and reduce the Ca2+ concentration necessary for muscle activation. To test this prediction, we have constructed and functionally characterized two troponin-C mutants (V45T and M48A mutations). Direct calcium-binding measurements in the mutants demonstrate an increase in the Ca2+ affinity for two low-affinity sites. Replacement of endogenous troponin C in skinned muscle fibers by TnC with mutations V45T or M48A increased the Ca2+ sensitivity of their tension development. These results show that the model can be used to construct mutants that regulate muscle contraction at lower Ca2+ concentrations. They provide further experimental support for the proposed calcium-induced conformational change of troponin C and suggest that the predicted transition plays a central role in the activation of the thin filament.

Two-dimensional 1H nuclear magnetic resonance studies of the half-saturated (Ca2+)1 state of calbindin D9k. Further implications for the molecular basis of cooperative Ca2+ binding.

J Mol Biol. 1993; 231: 415-30

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Calbindin D9k exhibits cooperative binding of two calcium ions, hence study of the half-saturated states of the protein is critical to understanding the binding process. However, the half-saturated states are not significantly populated under equilibrium conditions. To circumvent this problem, an absolutely conserved glutamic acid residue in the C-terminal binding site (site II) has been mutated to glutamine (E65Q), causing a substantial reduction in calcium affinity and permitting detailed two-dimensional 1H NMR analysis of calbindin D9k with a calcium ion bound only in the N-terminal EF-hand. Complete 1H resonance assignments have been obtained for (Ca2+)1 E65Q, as well as near complete assignments for the apo and (Ca2+)2 states. A value of 1.1(+/- 0.2) x 10(3) M-1 has been determined for the calcium binding constant in site II, from an analysis of the chemical shift changes in response to titration with calcium. The elements of secondary structure and global folding patterns were identified from nuclear Overhauser effects, backbone spin-spin coupling constants and the exchange rates of backbone amide protons. Although the mutation has only very small effects on the secondary structure and global fold of the protein, it so drastically lowers affinity for Ca2+ in the C-terminal site that (Ca2+)2 E65Q does not correspond to a standard (Ca2+)2 state. From the analysis of the half-saturated state, it is apparent that some reorganization of the structure and changes in the internal dynamics of calbindin D9k does occur for each step of the apo-->(Ca2+)1(I)-->(Ca2+)2 binding pathway. When the first ion is bound to the N-terminal EF-hand, that half of the molecule adopts a conformation and dynamic state similar to the fully calcium-loaded protein state, whereas only minor changes occur in the C-terminal EF-hand. It is only upon binding of the second calcium ion that the C-terminal EF-hand switches over to the fully calcium-loaded state. Together with the results from our earlier study of the apo-->(Ca2+)1(II)-->(Ca2+)2 binding pathway, these findings indicate that changes in protein conformation and dynamics associated with Ca2+ binding contribute to the observed positive cooperativity, and that the molecular details of the cooperative binding events are different for the two binding pathways.

A calculation strategy for the structure determination of symmetric dimers by 1H NMR.

Proteins. 1993; 17: 297-309

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The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter- and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving the ambiguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamics-based simulated annealing approach to minimize a target function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the method is useful both in situations where the structure of a homologous protein is known and in ab initio structure determination. The method can be extended to higher order symmetric multimers.

High-resolution three-dimensional structure of ribonuclease A in solution by nuclear magnetic resonance spectroscopy.

J Mol Biol. 1993; 229: 722-34

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High-resolution three-dimensional structures of bovine pancreatic ribonuclease A in aqueous solution have been determined by nuclear magnetic resonance (NMR) spectroscopy combined with restrained molecular dynamics calculations. The structures are based on: (1) 464 interproton distance constraints with accurate upper and lower limits, determined from build-up rates of nuclear Overhauser effects (NOE) by using the complete relaxation matrix; (2) 999 more approximate upper limits for interproton distances; and (3) 42 dihedral angle constraints (37 for phi and 5 for chi 1). A total of 16 structures were calculated, which show a root-mean-square (r.m.s.) deviation of 0.66 A for the backbone atoms and 1.68 A for all heavy-atoms. The converged structures are highly similar to those found in the crystal state. r.m.s. deviation of backbone atom positions in the crystal as compared to those in the average solution structure is 0.92 A. Observed differences are concentrated in loop regions and in the neighborhood of His119 and His48 side-chains. Dynamic aspects, such as H/D amide proton exchange and side-chain mobility have been examined.

The control of the formation of crystals in biological fluids is one of the most exciting field of research involving both organic and biochemical areas. Many organisms have evolved mechanisms which minimize or avoid the effects of nucleation and crystal growth formation. One of the most important mechanism is the interaction of specific proteins, called inhibitors, with crystals which alters their habits and leads to their elimination. This article, focused on saliva, pancreatic juice and bile, reviews our present knowledge on the structure-function relationships existing between these proteins and their ability to inhibit the growth of different calcium salt crystals.

Three-dimensional structure of the complex between acyl-coenzyme A binding protein and palmitoyl-coenzyme A.

J Mol Biol. 1993; 230: 1260-77

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Multidimensional 1H, 13C and 15N nuclear magnetic resonance spectroscopy has been used to study the complex between palmitoyl-coenzyme A and acyl-coenzyme A binding protein. The 1H and the 15N spectra of the holo-protein have been almost completely assigned and so has most of the 1H spectrum of the coenzyme A part of the protein-bound ligand. The palmitoyl part of the ligand has been uniformly labelled with 13C and the nuclear magnetic resonance signals of the carbon atoms and their protons have been assigned at the two ends of the hydrocarbon chain. A total of 1251 distance restraints from nuclear Overhauser effects and 131 dihedral angle restraints from three-bond coupling constants provided the basis for the structure calculation. A comparison of 20 structures calculated from these data to the average structure showed that they could be aligned with an atomic root-mean-square deviation of 1.3(+/- 0.2) A for all C, N, O, P and S atoms in protein and ligand. The apo-protein is a four-helix protein and this structure is maintained in the holo-protein. The four alpha-helices are Ac1 of residues 3 to 15, Ac2 from residue 20 to 36, Ac3 from 51 to 62, and Ac4 from 65 to 84. For the four alpha-helices of the peptide backbone of the holo-protein the root-mean-square deviation for the C, C alpha and N atoms was 0.42(+/- 0.08) A. The binding site for the palmitoyl-chain stretches between the N-terminal end of Ac3 where the carboxyl part binds, to the N-terminal of Ac3 where the omega-end of the palmitoyl part binds. The adenosine-3'-phosphate is bound near residues of each of the four helices in an arrangement where it can form salt bridges and/or hydrogen bonds to either backbone or side-chain atoms of Ala9, Tyr28, Lys32, Lys54 and Tyr73. The polar parts of the pantetheine and the pyrophosphate are structured in the bound ligand to form an interface with the solvent. Also the ligand forms a set of non-polar intramolecular interactions where the adenine, the pantetheine, and the palmitoyl-chain are associated, so overall the structure of the bound ligand seems to be organized to protect the lipophilic palmitoyl part from the polar solvent.

The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of approximately 15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three- and four-dimensional heteronuclear NMR spectroscopy. The structure is based on a total of 2973 experimental NMR restraints, comprising 2515 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.44 +/- 0.03 A for the backbone atoms, 0.83 +/- 0.03 A for all atoms, and 0.51 +/- 0.04 A for all atoms excluding disordered side chains. The N- and C-terminal residues (1-7 and 130-133, respectively) appear to be disordered. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The latter include a mini anti-parallel beta-sheet. A best fit superposition of the NMR structure of IL-4 with the 2.25 A resolution crystal structure [Wlodawer, A., Pavlovsky, A., & Gutschina, A. (1992) FEBS Lett. 309, 59-64] yields a backbone atomic rms difference of 1.37 A which can be mainly attributed to tighter packing of the helices in the crystal structure. This is indicated by an approximately 20% reduction in the axial separation of three pairs of helices (alpha A-alpha C, alpha A-alpha D, and alpha C-alpha D) in the crystal structure relative to the NMR structure and may reflect the greater flexibility of the molecule in solution which is reduced in the crystal due to intermolecular contacts. Comparison of the NMR structure of IL-4 with the X-ray structures of two other related proteins, granulocyte-macrophage colony stimulating factor [Diedrichs, K., Boone, T., & Karplus, P. A. (1992) Science 254, 1779-1782] and human growth hormone [de Vos, A. M., Ultsch, M., & Kossiakoff, A. A. (1992) Science 255, 306-312], that bind to the same hematopoietic superfamily of cell surface receptors reveals a remarkably similar topological fold, despite the absence of any significant overall sequence identity, and substantial differences in the relative lengths of the helices, the lengths and the nature of the various connecting elements, and the pattern and number of disulfide bridges.(ABSTRACT TRUNCATED AT 400 WORDS)

Calretinin is a member of the EF-hand calcium-binding protein family, with a high similarity with calbindin D28k. The chick calretinin cDNA sequence was reconstructed in a M13 vector and transferred into an expression plasmid derived from the pET series. The calretinin gene was expressed in Escherichia coli and produced immunoreactive calretinin of the expected size. Bacterially expressed calretinin was purified with successive ammonium-sulfate precipitation, DEAE chromatography, hydroxyapatite chromatography, Sephadex G-75 chromatography and Mono-Q chromatography. Normally, 1.0-1.5 mg calretinin was obtained from 1 l bacterial culture with a protein recovery of 0.5-1.5%. Calbindin D28k was purified similarly from bacteria using an expression plasmid provided by W. Hunziker. Calcium-binding activity of purified proteins was measured by equilibrium dialysis in calcium/EGTA mixtures with 45Ca as tracer. Both calretinin and calbindin D28k bound 3-4 Ca2+/molecule (calretinin, 4.0 +/- 0.5; calbindin D28k, 3.5 +/- 0.4), implying that at least one of the canonical EF-hand domains does not bind calcium. The Kd was 0.3-0.5 microM with little difference between the values for the two proteins.

A model for the calmodulin-peptide complex based on the troponin C crystal packing and its similarity to the NMR structure of the calmodulin-myosin light chain kinase peptide complex.

Protein Sci. 1993; 2: 620-5

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In the crystal structure of troponin C, the holo C-domain is bound in a head-to-tail fashion to the A-helix of the apo N-domain of a symmetry-related molecule. Using this interaction, we have proposed a model for the calmodulin-peptide complex. We find that the interaction of the C-domain with the A-helix is similar to that observed in the NMR structure of the calmodulin-myosin light chain kinase (MLCK) peptide complex. This similarity in binding has enabled us to make a precise sequence alignment of the target peptides in the calmodulin-binding cleft and to rationalize the amino acid sequence-dependent binding strengths of various peptides. Our model differs from that proposed by Strynadka and James (Proteins Struct. Funct. Genet. 7, 234-248, 1990) in that the peptides are rotated by 100 degrees in the calmodulin binding cleft.

Muscle fiber contraction is regulated through calcium-induced changes in the conformation of troponin C. In this study, we explored the relationship between the stability of a specific helix in the protein and the metal ion affinity of associated binding sites. Serial replacement of the amino acid at position 130 caused the calcium affinity of the paired Ca2+/Mg2+ sites to be attenuated. In the crystal structures of chicken and turkey troponin C, position 130 is the N-cap residue of the G-helix. The ion affinities of variant proteins were shifted in the order Ile < Gly < Asp < Asn < Thr < Ser. Although differing in ion affinities, the variant proteins all exhibited high cooperativity. The results of this study point to a specific relationship between alpha-helix stability and ion affinity in troponin C and suggest that troponin C may be a paradigm for protein folding problems.

Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

Biochemistry. 1993; 32: 9832-44

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The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kordel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site.

The aim of this work was to confirm the presence of calcylin in brain and to identify calcyclin positive cells. Calcyclin was identified in brain soluble proteins that were bound to phenyl-sepharose in a calcium dependent fashion. Specific antibodies against calcylin were found to stain subsets of brain neurones such as neurones in Ammons horn of hippocampus, granule cells in cerebellum, and neurones in brain stem. Glial cells which contain large amounts of S-100 beta protein were calcyclin negative. These results indicate that calcyclin is present in neurones, but not in glial cells.

Identification of the regions conferring calmodulin-like properties to troponin C.

J Biol Chem. 1993; 268: 11685-90

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The structural and functional correlations between troponin C (TnC) and calmodulin (CaM) were investigated by mutagenizing a synthetic cDNA coding rabbit skeletal muscle TnC. Compared with TnC, calmodulin lacks the N-terminal alpha-helical arm (N-helix), and its central helix is shorter due to the absence of 88KGK90 residues. Deleting both regions concomitantly (delta Nt delta KGK) elicited CaM-like regulation as tested (i) by smooth muscle contractility (maximal tension = 80 +/- 5% Po of control) and (ii) by the activation of phosphodiesterase (Vmax = 75 +/- 2% of control). The Ca(2+)-binding capacity of the mutant and the effect of the mutant on maximally Ca(2+)-activated tension of skinned rabbit psoas muscle fibers were both conserved. Furthermore, in the linker region of the central helix, replacing the TnC-characteristic 85EDAKGK90 successive residues with CaM-specific DTD residues generated a highly effective CaM mimic (Vmax = 96 +/- 2%) whether or not the N-helix was also retained. Apparent KD values (i.e. concentrations for half-maximal response) for the successful mutants were similar to each other but about 200-fold higher than that for CaM. A part of the alpha-helical linker region in CaM may unfold and bend to promote multiplicity of target interaction using all four hands (Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638; Meador, W. E., Means, A. R., and Quiocho, F. A. (1992) Science 257, 1251-1255). In contrast, our results suggest that the TnC central helix evolved to be less pliable by the combined influences of 85EDAKGK90 residues and the alpha-helical extension in N terminus, thereby keeping the N-terminal hands well separated from their C-terminal counterparts.

One- and two-dimensional NMR techniques were used to compare the structural consequences of Ca2+ binding to both the low and high affinity Ca2+ binding sites in recombinant cardiac troponin C (cTnC3). In the absence of Ca2+, the short beta-sheet located between the high affinity Ca2+/Mg2+ binding sites in the C-terminal domain was found to be absent or loosely formed as judged by the inter-residue NOEs and chemical shifts of resonances in the Ca2+ binding loops. In contrast, the N-terminal domain beta-sheet located between site II and the naturally inactive site I was present even in the absence of bound Ca2+. Calcium-binding mutant proteins having either an inactive Ca2+ binding site III (CBM-III) or an inactive Ca2+ binding site IV (CBM-IV) (Negele, J. C., Dotson, D., Liu, W., Sweeney, H. L., and Putkey, J. A. (1992) J. Biol. Chem. 267, 825-831) were used to study the structural consequences of Ca2+ binding to each of the high affinity sites located in the C-terminal domain. Only a single active Ca2+ binding site was found necessary for formation of the short beta-sheet between Ca2+ binding sites III and IV. However, the absence of bound Ca2+ at site III was found to produce greater instability in the C-terminal domain as judged from the mobility of the C-terminal aromatic hydrophobic cluster. Thus, Ca2+ binding to the high affinity sites in the C-terminal domain results in an ordering of the aromatic hydrophobic cluster, as well as formation of a short beta-sheet between Ca2+ binding sites III and IV. These results demonstrate that Ca2+ binding plays distinctive structural roles in the N- and C-terminal domains of cTnC.

The TR1C fragment of turkey skeletal muscle TnC (residues 12-87) comprises the two regulatory calcium binding sites of the protein. Complete assignments of the 1H-NMR resonances of the backbone and amino acid side chains of this domain in the absence of metal ions have been obtained using 2D 1H-NMR techniques. Sequential (i,i+1) and short-range (i,i+3) NOE connectivities define two helix-loop-helix calcium binding motifs, and long-range NOE connectivities indicate a short two-stranded beta-sheet formed between the two calcium binding loops. The two calcium binding sites are different in secondary structure. In terms of helix length, site II conforms to a standard "EF-hand" motif with the first helix ending one residue before the first calcium ligand and the second helix starting one residue after the beta-sheet. In site I, the first helix ends three residues before the first calcium ligand, and the second helix starts three residues after the beta-sheet. A number of long-range NOE connectivities between the helices define their relative orientation and indicate formation of a hydrophobic core between helices A, B, and D. The secondary structure and global fold of the TR1C fragment in solution in the calcium-free state are therefore very similar to those of the corresponding region in the crystal structure of turkey skeletal TnC [Herzberg, O., & James, M.N.G. (1988) J. Mol. Biol. 203, 761-779].

The structure of the 129-residue protein hen lysozyme has been determined in solution by two-dimensional 1H nuclear magnetic resonance methods. 1158 NOE distance restraints, and 68 phi and 24 chi 1 dihedral angle restraints were employed in conjunction with distance geometry and simulated annealing procedures. The overall C alpha root-mean-square deviation from the average for 16 calculated structures is 1.8(+/- 0.2) A, but excluding 14 residues in exposed disordered regions, this value reduces to 1.3(+/- 0.2) A. Regions of secondary structure, and the four alpha-helices in particular, are well defined (C alpha root-mean-square deviation 0.8(+/- 0.3) A for helices). The main-chain fold is closely similar to structures of the protein in the crystalline state. Furthermore, many of the internal side-chains are found in well-defined conformational states in the solution structures, and these correspond well with the conformational states found in the crystal. The general high level of definition of mainchain and many internal side-chains in the solution structures is reinforced by the results of an analysis of coupling constants and ring current shifts. Many side-chains on the surface, however, are highly disordered amongst the set of solution structures. In certain cases this disorder has been shown to be dynamic in origin by the examination of 3J alpha beta coupling constants.

The solution structures of mutant calbindin D9k's, as determined by NMR, show that the calcium-binding site can adopt different folds.

Biochemistry. 1993; 32: 8429-38

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The complete 1H NMR assignments have been obtained for five mutant proteins of calbindin D9k and the three-dimensional solution structures determined for two of the mutants. The structures have been determined using distance geometry and simulated annealing, with distance constraints from NMR. All mutants have modifications in the first calcium-binding site of calbindin (the N-terminal site designated the pseudo-EF-hand). The 3D structure of the mutant with the most extensive modifications in the pseudo-EF-hand shows that the site has turned inside-out and coordinates calcium as in the normal EF-hand (the C-terminal site). In a pseudo-EF-hand loop the calcium is coordinated by main-chain carbonyls, whereas calcium in the normal EF-hand is coordinated by side-chain carboxylates. The 3D structures and 1H NMR assignments show that in order to accomplish a change in the coordinating ligands of the pseudo-EF-hand the loop must be 12 residues long and have glycine in the sixth position. It does, however, seem possible to have alanine instead of aspartic acid in the first calcium coordinating position. The overall global fold of the proteins has not been affected by the mutations in the calcium-binding site, as compared to the wild-type calbindin D9k [Kordel, J., Skelton, N. J., Akke, M., & Chazin, W. J. (1993) J. Mol. Biol. (in press)]. The structures consist of two helix-calcium-binding loop-helix motifs, the so called EF-hands, and the loops are connected by a short antiparallel beta-sheet. All helices are pairwise in an antiparallel orientation.

The effect of recoverin-like calcium-binding proteins on the photoresponse of retinal rods.

Neuron. 1993; 10: 523-31

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The rod photoresponse is triggered by an enzyme cascade that stimulates cGMP hydrolysis. The resulting fall in cGMP leads to a decrease in Ca2+, which promotes photoresponse recovery by activating guanylate cyclase, causing cGMP resynthesis. In vitro biochemical studies suggest that Ca2+ activation of guanylate cyclase is medicated by recoverin, a 26 kd Ca(2+)-binding protein. To evaluate this, exogenous bovine recoverin and two other homologous Ca(2+)-binding proteins from chicken and Gecko retina were dialyzed into functionally intact Gecko rods using whole-cell recording. All three proteins prolonged the rising phase of the photoresponse without affecting the kinetics of response recovery. These results suggest that recoverin-like proteins affect termination of the transduction cascade, rather than mediate Ca(2+)-sensitive activation of guanylate cyclase.

Critical minimum length of the central helix in troponin C for the Ca2+ switch in muscular contraction.

J Biol Chem. 1993; 268: 19232-8

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In the troponin C (TnC) dumbbell, the NH2- and COOH-terminal lobes are well delineated, but the role of the central helix and especially the function of its long length remain doubtful. To study this, we used a cDNA construct encoding rabbit fast-twitch muscle TnC, comprising multiple restriction sequences to facilitate mutagenesis (Babu, A., Su, H., Ryu, Y. & Gulati, J. (1992) J. Biol. Chem. 267, 15469-15474). Systematically, we have deleted 3-12 amino acid residues from the central helix and examined their effects in maximally activated skinned muscle fibers. Limiting the deletions to 7 amino acid residues manifested little change in maximal force development (Sheng, Z., Francois, J. M., Hitchcock, S. E. & Potter, J. D. (1991) J. Biol. Chem. 266, 5711-5715). However, with further deletions, we now find that contractility was inhibited pari passu; by 12 deletions, the inhibition was complete. The critical minimum length for the central helix is thereby estimated as 27 A. The Ca2+ binding capacity (4 mol of Ca2+/mol of protein) as well as the structural characteristics (alpha-helicity by CD measurements and the fluorescence emitted by Tyr-109) indicated a well preserved global conformation of the short mutant. However, surprisingly, two of these short mutants filled each TnC slot under highly specific superloading conditions: one short molecule was taken up in EGTA solution, and the second molecule was captured and retained with Ca2+. They also rescued the contractile switch, evidently in a bimolecular reaction. Another short variant (putative skeletal fast muscle TnC-I-II), in which the NH2-terminal Ca(2+)-binding sites were incapacitated, failed to respond to superloading, indicating that sites III and IV could not substitute for sites I and II. The results suggest that a critical role of the central helix linker in TnC is to keep the two lobes optimally apart, evidently in proximity of their respective target sites on troponin I in the fiber.

The effect of decreased protein flexibility on the stability and calcium binding properties of calbindin D9k has been addressed in studies of a disulfide bridged calbindin D9k mutant, denoted (L39C + P43M + I73C), with substitutions Leu 39-->Cys, Ile 73-->Cys, and Pro 43-->Met. Backbone 1H NMR assignments show that the disulfide bond, which forms spontaneously under air oxidation, is well accommodated. The disulfide is inserted on the opposite end of the protein molecule with respect to the calcium sites, to avoid direct interference with these sites, as confirmed by 113Cd NMR. The effect of the disulfide bond on calcium binding was assessed by titrations in the presence of a chromophoric chelator. A small but significant effect on the cooperativity was found, as well as a very modest reduction in calcium affinity. The disulfide bond increases Tm, the transition midpoint of thermal denaturation, of calcium free calbindin D9k from 85 to 95 degrees C and Cm, the urea concentration of half denaturation, from 5.3 to 8.0 M. Calbindins with one covalent bond linking the two EF-hand subdomains are equally stable regardless if the covalent link is the 43-44 peptide bond or the disulfide bond. Kinetic remixing experiments show that separated CNBr fragments of (L39C + P43M + I73C), each comprising one EF-hand, form disulfide linked homodimers. Each homodimer binds two calcium ions with positive co-operativity, and an average affinity of 10(6) M-1. Disulfide linkage dramatically increases the stability of each homodimer. For the homodimer of the C-terminal fragment Tm increases from 59 +/- 2 without covalent linkage to 91 +/- 2 degrees C with disulfide, and Cm from approximately 1.5 to 7.5 M. The overall topology of this homodimer is derived from 1H NMR assignments and a few key NOEs.

In the first report in this series we presented dendrograms based on 152 individual proteins of the EF-hand family. In the second we used sequences from 228 proteins, containing 835 domains, and showed that eight of the 29 subfamilies are congruent and that the EF-hand domains of the remaining 21 subfamilies have diverse evolutionary histories. In this study we have computed dendrograms within and among the EF-hand subfamilies using the encoding DNA sequences. In most instances the dendrograms based on protein and on DNA sequences are very similar. Significant differences between protein and DNA trees for calmodulin remain unexplained. In our fourth report we evaluate the sequences and the distribution of introns within the EF-hand family and conclude that exon shuffling did not play a significant role in its evolution.

In the previous three reports in this series we demonstrated that the EF-hand family of proteins evolved by a complex pattern of gene duplication, transposition, and splicing. The dendrograms based on exon sequences are nearly identical to those based on protein sequences for troponin C, the essential light chain myosin, the regulatory light chain, and calpain. This validates both the computational methods and the dendrograms for these subfamilies. The proposal of congruence for calmodulin, troponin C, essential light chain, and regulatory light chain was confirmed. There are, however, significant differences in the calmodulin dendrograms computed from DNA and from protein sequences. In this study we find that introns are distributed throughout the EF-hand domain and the interdomain regions. Further, dendrograms based on intron type and distribution bear little resemblance to those based on protein or on DNA sequences. We conclude that introns are inserted, and probably deleted, with relatively high frequency. Further, in the EF-hand family exons do not correspond to structural domains and exon shuffling played little if any role in the evolution of this widely distributed homolog family. Calmodulin has had a turbulent evolution. Its dendrograms based on protein sequence, exon sequence, 3'-tail sequence, intron sequences, and intron positions all show significant differences.

The chromosomal assignments of genes belonging to the EF-hand family which have a common origin are compiled in this article. So far data are available from 27 human gene loci belonging to 6 subfamilies and 8 murine loci belonging to 4 subfamilies. Chromosomal localization has been obtained by somatic-cell hybrid analysis using the Southern blot technique or PCR amplification, metaphase spread in situ hybridization, or isolation of the particular genes from chromosome-specific libraries. Except for genes of the S-100 alpha proteins which are grouped on human chromosome 1q12-25 and mouse chromosome 3, no linkage has been found for genes encoding EF-hand proteins, indicating absence of selective pressure for maintaining chromosomal clustering. Six of these genes map to known syntenic groups conserved in the human and mouse genomes. This suggests that chromosomal translocations occurred before divergence of these species. The possible significance of chromosomal positioning with respect to nearby located known genes and genetic disease loci is discussed.

The structure in solution of crambin, a small protein of 46 residues, has been determined from 2D NMR data using an iterative relaxation matrix approach (IRMA) together with distance geometry, distance bound driven dynamics, molecular dynamics, and energy minimization. A new protocol based on an "ensemble" approach is proposed and compared to the more standard initial rate analysis approach and a "single structure" relaxation matrix approach. The effects of fast local motions are included and R-factor calculations are performed on NOE build-ups to describe the quality of agreement between theory and experiment. A new method for stereospecific assignment of prochiral groups, based on a comparison of theoretical and experimental NOE intensities, has been applied. The solution structure of crambin could be determined with a precision (rmsd from the average structure) of 0.7 A on backbone atoms and 1.1 A on all heavy atoms and is largely similar to the crystal structure with a small difference observed in the position of the side chain of Tyr-29 which is determined in solution by both J-coupling and NOE data. Regions of higher structural variability (suggesting higher mobility) are found in the solution structure, in particular for the loop between the two helices (Gly-20 to Pro-22).

A novel peptide designed for sensitization of terbium (III) luminescence.

FEBS Lett. 1993; 333: 96-8

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Several synthetic peptides, modelled from a Ca(2+)-binding loop of the EF-hand family of proteins, were prepared containing cysteine residues. The peptide, GDKNADGFICFEEL, was labelled covalently at the cysteine residue (loop position 9) with iodoacetamidosalicylic acid. This novel conjugate is a metal-binding loop containing a salicylic acid side chain that could not only chelate Tb3+ in conjunction with the other chelating groups in the sequence, but could also sensitize Tb3+ luminescence. The loop had a high Tb3+ affinity, with stoichiometric binding observed under experimental conditions. The luminescence from the Tb(3+)-peptide complex was more than 10-fold greater than the luminescence reported from a related peptide which contained Trp as the Tb3+ donor at loop position 7. This peptide has significant potential for use in lanthanide-based time-resolved luminescence immunoassays.

The calcium-binding proteins MRP8 and MRP14 form a membrane-associated heterodimer in a subset of monocytes/macrophages present in acute but absent in chronic inflammatory lesions.

Eur J Immunol. 1992; 22: 1891-7

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Monocytes/macrophages expressing an epitope recognized by a monoclonal antibody 27E10 are present in acute but are absent in chronic inflammatory disorders. This report shows that the 27E10 antigen is formed by noncovalent association of the two Ca(2+)-binding proteins MRP8 and MRP14 which belong to the S100 protein family. Identification has been confirmed immunochemically, by matrix-assisted UV-laser desorption/ionization spectrometry and by partial amino acid sequencing. Surface expression of the MRP8/MRP14 complex on a subset of monocytes is reported for the first time and shown to be up-regulated in a Ca(2+)-dependent manner. The 27E10 surface-positive monocytes isolated by cell separation techniques release high amounts of tumor necrosis factor-alpha and interleukin-1 beta in contrast to their 27E10 surface-negative counterparts thus emphasizing their role in inflammation.

Translocation of a small cytosolic calcium-binding protein (MRP-8) to plasma membrane correlates with human neutrophil activation.

J Biol Chem. 1992; 267: 19379-82

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To further understand the mechanisms involved in phagocyte activation in general and in NADPH oxidase activation in particular, a polyclonal antibody was raised in rabbit against a partially purified oxidase preparation. The enzyme was solubilized from zymosan-activated human neutrophils and resting cells and separated by preparative isoelectric focusing electrophoresis. A polyclonal antibody was raised in rabbit against the pI 5.0 fraction, which had the maximum superoxide-producing capacity. Analysis of the polyclonal antibody revealed marked differences between activated and resting neutrophils. The antibody recognized in particular an 8-kDa protein (p8) in resting human neutrophil cytosol and in the membrane of zymosan-activated cells. A polyclonal antibody (anti-p8) was raised against the pure cytosolic p8 protein. This anti-p8 reacted not only with p8, but also with cytosolic proteins of 14 kDa and 6 kDa. N-terminal amino acid sequence analysis of p8 revealed homology with the calcium-binding myeloid related protein (MRP-8). Upon neutrophil activation, translocation of the 8- and 14-kDa proteins to the membrane was observed with stimuli known to depend on extracellular calcium. In calcium-depleted medium, the absence of translocation correlated with a depression of superoxide production, supporting a role for the calcium-binding protein in cellular activation.

Role of facilitated diffusion of calcium by calbindin in intestinal calcium absorption.

Am J Physiol. 1992; 262: 51726-51726

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Computer simulations of transcellular Ca2+ transport in enterocytes were carried out using the simulation program SPICE. The program incorporated a negative-feedback entry of Ca2+ at the brush-border membrane that was characterized by an inhibitor constant of 0.5 microM cytosolic Ca2+ concentration ([Ca2+]). The basolateral Ca(2+)-ATPase was simulated by a four-step mechanism that resulted in Michaelis-Menten kinetics with a Michaelis constant of 0.24 microM [Ca2+]. The cytosolic diffusion of Ca2+ was simulated by dividing the cytosol into 10 slabs of equal width. Ca2+ binding to calbindin-D9K was simulated in each slab, and diffusion of free Ca2+, free calbindin, and Ca(2+)-laden calbindin was simulated between each slab. The cytosolic [Ca2+] of the simulated cells was regulated within the physiological range. Calbindin-D9K reduced the cytosolic [Ca2+] gradient, increased Ca2+ entry into the cell by removing the negative-feedback inhibition of Ca2+ entry, increased cytosolic Ca2+ flow, and increased the efflux of Ca2+ across the basolateral membrane by increasing the free [Ca2+] immediately adjacent to the pump. The enhancement of transcellular Ca2+ transport was nearly linearly dependent on calbindin-D9K concentration. The values of the dissociation constant (Kd) for calbindin-D9K were previously obtained experimentally in the presence and absence of KCl. Calbindin with the Kd obtained in the presence of KCl enhanced the simulated Ca2+ transport more than with the Kd obtained in the absence of KCl. This result suggests that the physiological Kd of calbindin is optimal for the enhancement of transcellular Ca2+ transport. The simulated Ca2+ flow was less than that predicted from the "near-equilibrium" analytic solution of the reaction-diffusion problem.

CAP-50, a calcyclin-associated protein with an apparent molecular mass of 50 kDa, was purified and proved to be a novel annexin [Tokumitsu, H. et al. (1992) J. Biol. Chem. 267, 8919-8924]. We examined the binding of CAP-50 to other Ca(2+)-binding proteins which have two of four EF-hand structures, by a co-precipitation assay with phospholipid (phosphatidylserine). Among nine Ca(2+)-binding proteins (calcyclin, S-100 proteins, p11, calgizzarin, calvasculin, calmodulin and troponin C) examined, only calcyclin interacted with CAP-50. These results clearly show that the interaction of CAP-50 to calcyclin is specific, i.e. other Ca(2+)-binding proteins with the EF-hand structure could not substitute for calcyclin, thereby suggesting the possible role in specific regulation of the function of CAP-50 by Ca2+/calcyclin.

Stoichiometry of calcium binding to a synthetic heterodimeric troponin-C domain.

Biopolymers. 1992; 32: 391-7

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In this work we describe calcium binding to two synthetic 34-residue peptides, determined by 1H-nmr spectroscopy. The peptides investigated, SCIII and SCIV, encompass the calcium-binding sites III and IV, respectively, of troponin-C. In the absence of calcium it has previously been shown that each of these peptides possesses little regular secondary structure. Further, the 1H-nmr spectra of an equimolar mixture of both of these apo-peptides (apo-SCIII/SCIV) shows that little interaction occurs between peptides. Upon calcium binding the spectral changes that occur to SCIII/SCIV are consistent with global conformational changes in both peptides. We have shown previously that these conformational changes are a product of calcium binding to SCIII and SCIV to form a two-site heterodimer Ca2-SCIII/SCIV. It is proposed that this calcium-induced folding proceeds via calcium binding to SCIII to form Ca-SCIII, peptide association with apo-SCIV to form the heterodimer Ca-SCIII/SCIV, and calcium binding to form Ca2-SCIII/SCIV. The dissociation constants involved in this pathway, K1, Kd, and K2, respectively, have been determined by stoichiometric calcium titration of SCIII/SCIV, monitored by 1H-nmr spectroscopy. Using this procedure it has been determined that K1 = 3 microM, Kd = 10 microM, and K2 = 2 microM.

In the first report in this series we described the relationships and evolution of 152 individual proteins of the EF-hand subfamilies. Here we add 66 additional proteins and define eight (CDC, TPNV, CLNB, LPS, DGK, 1F8, VIS, TCBP) new subfamilies and seven (CAL, SQUD, CDPK, EFH5, TPP, LAV, CRGP) new unique proteins, which we assume represent new subfamilies. The main focus of this study is the classification of individual EF-hand domains. Five subfamilies--calmodulin, troponin C, essential light chain, regulatory light chain, CDC31/caltractin--and three uniques--call, squidulin, and calcium-dependent protein kinase--are congruent in that all evolved from a common four-domain precursor. In contrast calpain and sarcoplasmic calcium-binding protein (SARC) each evolved from its own one-domain precursor. The remaining 19 subfamilies and uniques appear to have evolved by translocation and splicing of genes encoding the EF-hand domains that were precursors to the congruent eight and to calpain and to SARC. The rates of evolution of the EF-hand domains are slower following formation of the subfamilies and establishment of their functions. Subfamilies are not readily classified by patterns of calcium coordination, interdomain linker stability, and glycine and proline distribution. There are many homoplasies indicating that similar variants of the EF-hand evolved by independent pathways.

1H-NMR spectroscopy is employed to study the interaction between rabbit skeletal muscle troponin (C (TnC) and wasp venom tetradecapeptide mastoparan. We monitored the spectral change of the following species of TnC as a function of mastoparan concentration: apoTnC, Ca(2+)-saturated TnC (Ca4TnC) and Ca(2+)-half loaded TnC (Ca2TnC). When apo-TnC is titrated with mastoparan, line-broadening is observed for the ring-current shifted resonance of Phe-23, Ile-34, Val-62 and Phe-72 and the downfield-shifted CH alpha-resonances of Asp-33, Thr-69 and Asp-71; these residues are located in the N-domain. When Ca4TnC is titrated with mastoparan, chemical shift change is observed for the ring-current shifted resonances of Phe-99, Ile-110 and Phe-148 and the downfield-shifted CH alpha-resonances of Asn-105, Ala-106, Ile-110 and Ile-146 and aromatic resonance of Tyr-109 and His-125; these residues are located in the C-domain. The resonance of Phe-23, Asp-33, Asp-71, Phe-72, Phe-99, Tyr-109, Ile-146, His-125 and Phe-148 in both N- and C-domains changes when Ca2TnC is titrated with mastoparan. These results suggest that mastoparan binds to the N-domain of apo-TnC, the C-domain of Ca4TnC and the N- and C-domains of Ca2TnC; the hydrophobic cluster in each domain is involved in binding. As mastoparan binds to TnC, the above resonances shift to their normal chemical shift positions. The stability of the cluster and the beta-sheet is reduced by mastoparan-binding. These results suggest that the conformation of the hydrophobic cluster and the neighboring beta-sheet change to a loose form. The stability of the N-domain of Ca2TnC and Ca4TnC increases when these species bind 1 mol of mastoparan at the C-domain. These results suggest a mastoparan-induced interaction between the N- and C-domains of TnC.

Molecular cloning and characterization of grancalcin, a novel EF-hand calcium-binding protein abundant in neutrophils and monocytes.

J Biol Chem. 1992; 267: 2928-33

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A novel EF-hand Ca(2+)-binding protein we have called grancalcin has been identified and characterized. This protein is particularly abundant in neutrophils and monocytes, with relatively small amounts in lymphocytes. The cDNA for this protein has been cloned and sequenced. The sequence predicts that the protein is composed of 217 amino acids, with a molecular mass of 24,010 daltons. It contains four EF-hand calcium-binding motifs and exhibits strong homology to sorcin, one of two proteins overexpressed in multidrug-resistant cells whose function is unknown. There are potentially one phosphorylation and two glycosylation sites. The 1.65-kilobase mRNA is detected in bone marrow and is present in neutrophils, monocytes, macrophages, B and T lymphocytes, and the promyelocytic cell line HL60s. The protein displays a Ca(2+)-dependent translocation to the granules and plasma membrane of neutrophils, suggesting that it might play an effector role in the specialized functions of these cells.

The three-dimensional solution structure of native, intact porcine calbindin D9k has been determined by distance geometry and restrained molecular dynamics calculations using distance and dihedral angle constraints obtained from 1H NMR spectroscopy. The protein has a well-defined global fold consisting of four helices oriented in a pairwise antiparallel manner such that two pairs of helix-loop-helix motifs (EF-hands) are joined by a linker segment. The two EF-hands are further coupled through a short beta-type interaction between the two Ca(2+)-binding loops. Overall, the structure is very similar to that of the highly homologous native, minor A form of bovine calbindin D9k determined by X-ray crystallography [Szebenyi, D. M. E., & Moffat, K. (1986) J. Biol. Chem. 261, 8761-8776]. A model structure built from the bovine calbindin D9k crystal structure shows several deviations larger than 2 A from the experimental distance constraints for the porcine protein. These structural differences are efficiently removed by subjecting the model structure to the experimental distance and dihedral angle constraints in a restrained molecular dynamics protocol, thereby generating a model that is very similar to the refined distance geometry derived structures. The N-terminal residues of the intact protein that are absent in the minor A form appear to be highly flexible and do not influence the structure of other regions of the protein. This result is important because it validates the conclusions drawn from the wide range of studies that have been carried out on minor A forms rather than the intact calbindin D9k.

VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain.

Brain Res Mol Brain Res. 1992; 15: 133-40

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Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca(2+)-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca(2+)-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca(2+)-dependent manner, are discussed.

Dissection of calbindin D9k into two Ca(2+)-binding subdomains by a combination of mutagenesis and chemical cleavage.

FEBS Lett. 1992; 298: 211-4

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Calbindin D9k is a 75-residue globular protein made up of two Ca2+ binding subdomains of the EF-hand type. In order to examine the subdomains independently, a method was devised to selectively cleave the loop between them. Using site-directed mutagenesis, a unique methionine was substituted for Pro43 in the loop, thus allowing cleavage using cyanogen bromide. Agarose gel electrophoresis shows that the fragments have a high affinity for one another, although less so in the absence of calcium. 1H-NMR spectra of the fragments indicate that the structures of the heterodimers are changed little from that of the intact protein. However, the Ca2+ binding constants of the individual subdomains are several orders of magnitude lower than for the corresponding sites in the uncleaved protein.

Structure of a sarcoplasmic calcium-binding protein from Nereis diversicolor refined at 2.0 A resolution.

J Mol Biol. 1992; 224: 413-26

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The crystal structure of a sarcoplasmic Ca(2+)-binding protein (SCP) from the sandworm Nereis diversicolor has been determined and refined at 2.0 A resolution using restrained least-squares techniques. The two molecules in the crystallographic asymmetric unit, which are related by a non-crystallographic 2-fold axis, were refined independently. The refined model includes all 174 residues and three calcium ions for each molecule, as well as 213 water molecules. The root-mean-square difference in co-ordinates for backbone atoms and calcium ions of the two molecules is 0.51 A. The final crystallographic R-factor, based on 18,959 reflections in the range 2.0 A less than or equal to d less than or equal to 7.0 A, with intensities exceeding 2.0 sigma, is 0.182. Bond lengths and bond angles in the molecules have root-mean-square deviations from ideal values of 0.013 A and 2.2 degrees, respectively. SCP has four distinct domains with the typical helix-loop-helix (EF-hand) Ca(2+)-binding motif, although the second Ca(2+)-binding domain is not functional due to amino acid changes in the loop. The structure shows several unique features compared to other Ca(2+)-binding proteins with four EF-hand domains. The overall structure is highly compact and globular with a predominant hydrophobic core, unlike the extended dumbbell-shaped structure of calmodulin or troponin C. A hydrophobic tail at the COOH terminus adds to the structural stability by packing against a hydrophobic pocket created by the folding of the NH2 and COOH-terminal Ca(2+)-binding domain pairs. The first and second domains show different helix-packing arrangements from any previously described for Ca(2+)-binding proteins.

An efficient automated computer vision based technique for detection of three dimensional structural motifs in proteins.

J Biomol Struct Dyn. 1992; 9: 769-89

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As the number of available three dimensional coordinates of proteins increases, it is now recognized that proteins from different families and topologies are constructed from independent motifs. Detection of specific structural motifs within proteins aids in understanding their role and the mechanism of their operation. To aid in identification and use of these motifs it has become necessary to develop efficient methods for systematic scanning of structural databases. To date, methods of structural protein comparison suffer from at least one of the following limitations: (1) are not fully automated (require human intervention), (2) are limited to relatively similar structures, (3) are constrained to linear alignments of the structures, (4) are sensitive to insertions, deletions or gaps in the sequences or (5) are very time consuming. We present a method to overcome the above limitations. The method discovers and ranks every piece of structural similarity between the structures compared, thus allowing the simultaneous detection of real 3-D motifs in different domains, between domains, in active sites, surfaces etc. The method uses the Geometric Hashing Paradigm which is an efficient technique originally developed for Computer Vision. The algorithm exploits the geometrical constraints of rigid objects, it is especially geared towards recognition of partial structures in rigid objects belonging to large data bases and is straightforwardly parallelizable. Computer Vision techniques are for the first time applied to molecular structure comparison, resulting in an efficient, fully automated tool. The method has been tested in a number of cases, including comparisons of the haemoglobins, immunoglobulins, serine proteinases, calcium binding proteins, DNA binding proteins and others. In all examples our results were equivalent to the published results from previous methods and in some cases additional structural information was obtained by our method.

Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2+ signal pathway of the nervous system.

The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites. This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions. Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5). Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.

Crystallographic studies of a calcium binding lysozyme from equine milk at 2.5 A resolution.

J Biochem (Tokyo). 1992; 111: 141-3

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The crystal structure of a calcium binding equine lysozyme has been determined at 2.5 A resolution by means of molecular replacement. The energy minimized equine lysozyme as the starting model, was refined with the molecular dynamics program, X-PLOR, and the R factor of the current model was found to be 24% without any water molecules. The conformation of the calcium binding loop is similar to that of alpha-lactalbumin. The profiles of backbone atomic displacements throughout the lysozyme and alpha-lactalbumin superfamilies are comparable as well as their homologous tertiary structures.

S100 beta is a calcium-binding protein with neurotrophic and mitogenic activities, both of which may be mediated by the protein's ability to stimulate an increase in intracellular free calcium ([Ca2+]i). These extracellular trophic activities of S100 beta require a disulfide-linked, dimeric form of the protein. In this chapter, we present a minireview on the current state of knowledge concerning extracellular functions of S100 beta, with emphasis on the potential relevance of these activities to neuropathological disorders. We also report a simplified procedure for preparation of pharmacological amounts of biologically active S100 beta dimers, based on the finding that formation of disulfide-linked S100 beta dimers can be stimulated by the presence of calcium or lipid.

Nuclear magnetic resonance studies of the internal dynamics in Apo, (Cd2+)1 and (Ca2+)2 calbindin D9k. The rates of amide proton exchange with solvent.

J Mol Biol. 1992; 227: 1100-17

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The backbone dynamics of the EF-hand Ca(2+)-binding protein, calbindin D9k, has been investigated in the apo, (Cd2+)1 and (Ca2+)2 states by measuring the rate constants for amide proton exchange with solvent. 15N-1H correlation spectroscopy was utilized to follow direct 1H-->2H exchange of the slowly exchanging amide protons and to follow indirect proton exchange via saturation transfer from water to the rapidly exchanging amide protons. Plots of experimental rate constants versus intrinsic rate constants have been analyzed to give qualitative insight into the opening modes of the protein that lead to exchange. These results have been interpreted within the context of a progressive unfolding model, wherein hydrophobic interactions and metal chelation serve to anchor portions of the protein, thereby damping fluctuations and retarding amide proton exchange. The addition of Ca2+ or Cd2+ was found to retard the exchange of many amide protons observed to be in hydrogen-bonding environments in the crystal structure of the (Ca2+)2 state, but not of those amide protons that were not involved in hydrogen bonds. The largest changes in rate constant occur for residues in the ion-binding loops, with substantial effects also found for the adjacent residues in helices I, II and III, but not helix IV. The results are consistent with a reorganization of the hydrogen-bonding networks in the metal ion-binding loops, accompanied by a change in the conformation of helix IV, as metal ions are chelated. Further analysis of the results obtained for the three states of metal occupancy provides insight into the nature of the changes in conformational fluctuations induced by ion binding.

Backbone dynamics of calcium-loaded calbindin D9k have been investigated by two-dimensional proton-detected heteronuclear nuclear magnetic resonance spectroscopy, using a uniformly 15N enriched protein sample. Spin-lattice relaxation rate constants, spin-spin relaxation rate constants, and steady-state [1H]-15N nuclear Overhauser effects were determined for 71 of the 72 backbone amide 15N nuclei. The relaxation parameters were analyzed using a model-free formalism that incorporates the overall rotational correlation time of the molecule, and a generalized order parameter (S2) and an effective internal correlation time for each amide group. Calbindin D9k contains two helix-loop-helix motifs joined by a linker loop at one end of the protein and a beta-type interaction between the two calcium-binding loops at the other end. The amplitude of motions for the calcium-binding loops and the helices are similar, as judged from the average S2 values of 0.83 +/- 0.05 and 0.85 +/- 0.04, respectively. The linker region joining the two calcium-binding subdomains of the molecule has a significantly higher flexibility, as indicated by a substantially lower average S2 value of 0.59 +/- 0.23. For residues in the linker loop and at the C-terminus, the order parameter is further decomposed into separate order parameters for motional processes on two distinct time scales. The effective correlation times are significantly longer for helices I and IV than for helices II and III or for the calcium-binding loops. Residue by residue comparisons reveal correlations of the order parameters with both the crystallographic B-factors and amide proton exchange rates, despite vast differences in the time scales to which these properties are sensitive. The order parameters are also utilized to distinguish regions of the NMR-derived three-dimensional structure of calbindin D9k that are poorly defined due to inherently high flexibility, from poorly defined regions with average flexibility but a low density of structural constraints.

The glial-derived protein S100 beta can act as a mitogen or a neurotrophic factor, stimulating proliferation of glial cells or differentiation of immature neurons. We report here that dimeric S100 beta evokes increases in intracellular free calcium concentrations ([Ca2+]i) in both glial cells and neuronal cells. The [Ca2+]i increase exhibited a rapid transient component which was not affected by removal of extracellular calcium and a sustained component which appeared to require influx of extracellular calcium through Ni(2+)-sensitive channels. S100 beta also stimulated hydrolysis of phosphoinositides, suggesting a mobilization of calcium from intracellular stores. These data suggest that although the final biological responses of neuronal and glial cells to S100 beta are different, transduction of the S100 beta signal in both cell types involves changes in [Ca2+]i.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.

The three-dimensional solution structure of apo-neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined beta-sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of apo-neocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven-stranded antiparallel beta-barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo-protein complex and for the chromophore specificity within the antitumoral family.

The distribution of calcyclin in human tissues was studied using polyclonal antibodies against this protein. In all organs examined (breast, heart, intestine, kidney, liver, ovary, placenta, stomach, thymus, and uterus) only epithelial cells and fibroblasts were stained. This suggests that calcyclin expression is related either to proliferation rate or secretion activity. The data show that calcyclin might be considered as a marker of some human epithelial cells and fibroblasts.

The three-dimensional structure of the N-terminal 51-residue domain of recombinant hirudin in aqueous solution was determined by 1H nuclear magnetic resonance (NMR) spectroscopy, and the resulting high-quality solution structure was compared with corresponding structures obtained from studies with the intact, 65-residue polypeptide chain of hirudin. On the basis of 580 distance constraints derived from nuclear Overhauser effects and 109 dihedral angle constraints, a group of 20 conformers representing the solution structure of hirudin(1-51) was computed with the program DIANA and energy-minimized with a modified version of the program AMBER. Residues 3 to 30 and 37 to 48 form a well-defined molecular core with two antiparallel beta-sheets composed of residues 14 to 16 and 20 to 22, and 27 to 31 and 36 to 40, and three reverse turns at residues 8 to 11 (type II), 17 to 20 (type II') and 23 to 26 (type II). The average root-mean-square deviation of the individual NMR conformers relative to their mean co-ordinates is 0.38 A for the backbone atoms and 0.77 A for all heavy atoms of these residues. Increased structural disorder was found for the N-terminal dipeptide segment, the loop at residues 31 to 36, and the C-terminal tripeptide segment. The solution structure of hirudin(1-51) has the same molecular architecture as the corresponding polypeptide segment in natural hirudin and recombinant desulfatohirudin. It is also closely similar to the crystal structure of the N-terminal 51-residue segment of hirudin in a hirudin-thrombin complex, with root-mean-square deviations of the crystal structure relative to the mean solution structure of 0.61 A for the backbone atoms and 0.91 A for all heavy atoms of residues 3 to 30 and 37 to 48. Further coincidence is found for the loop formed by residues 31 to 36, which shows increased structural disorder in all available solution structures of hirudin, and of which residues 32 to 35 are not observable in the electron density map of the thrombin complex. Significant local structural differences between hirudin(1-51) in solution and hirudin in the crystalline thrombin complex were identified mainly for the N-terminal tripeptide segment and residues 17 to 21. These are further analyzed in an accompanying paper.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectroscopic analysis of a methionine-48 to tyrosine mutant of chicken troponin C.

Biochemistry. 1992; 31: 9703-8

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A mutant (M48Y) of chicken skeletal muscle troponin C was prepared in which Tyr replaced Met-48 of the recombinant protein (rTnC). Since Tyr and Trp are normally absent, spectral properties could be unambiguously assigned to the site of substitution. In the crystal structure, this residue lies at the COOH-terminal end of the B-helix of the N domain in a region postulated to undergo a significant conformational change to a more polar environment upon Ca2+ binding [Herzberg et al. (1986) J. Biol. Chem. 261, 2638-2644]. Comparison of the far-UV CD spectra of M48Y and rTnC in the absence and presence of Ca2+ indicated no overall structural alteration due to the mutation. However, Ca2+ titration of the ellipticity change showed a reduction in Ca2+ affinity and cooperativity of sites I and II. A Ca(2+)-induced increase in the near-UV ellipticity of M48Y at pH 7.12 and a red shift in its UV absorbance spectrum occurred over a range of free [Ca2+] attributable to the N-domain transition only. This was largely abolished at pH 5.3 where Ca2+ no longer binds to sites I and II. That region of the 1H NMR spectrum attributable to Tyr was broadened upon Ca2+ binding. These Ca(2+)-induced changes are consistent with the environment of the Tyr side chain becoming chiral, less polar, and more immobile, all in a direction opposite to that predicted. These observations indicate that while the general features of the postulated model are valid, it is unlikely to be correct in detail.

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

Protein Sci. 1992; 1: 1595-603

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The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.

Calcium binding to brain and erythrocyte spectrins was studied at physiological ionic strength by a calcium overlay assay and aqueous two-phase partitioning. When the spectrins were immobilized on nylon membranes by slot blotting, the overlay assay showed that even though both spectrins bound 45Ca2+, the brain protein displayed much greater affinity for calcium ions than erythrocyte spectrin did. Since the observed binding was weaker than that displayed by calmodulin under similar conditions, the overlay assay results indicated that the binding must be weaker than 1 microM. The phase partition experiments showed that there are at least two sites for calcium on brain spectrin and that calcium binding to one of these sites is reduced significantly by magnesium ions. From the partition isotherm, the dissociation constants were estimated as 50 microM for the Mg(2+)-independent site and 150 microM for the Mg(2+)-dependent site. The phase partition results also showed that erythrocyte spectrin bound calcium ions at least 1 order of magnitude weaker. By examining calcium binding to slot-blotted synthetic peptides, we identified two binding sites in brain spectrin. One mapped to the second putative calcium binding site (EF-hand) in alpha-spectrin and the other to the 36 amino acid residue long insert in domain 11. In addition, a tryptic fragment derived from the C-terminal of erythrocyte alpha-spectrin, which contained the two postulated EF-hands, also bound calcium. These findings suggest that the calcium signal system may also involve direct binding of calcium to spectrin beside known calcium modulators such as calmodulin and calpain.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the solution structure of a synthetic two-site calcium-binding homodimeric protein domain by NMR spectroscopy.

Biochemistry. 1992; 31: 9572-80

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The solution structure of a 34-residue synthetic calcium-binding peptide from site III of chicken troponin-C has been determined by 1H NMR spectroscopy. In solution and in the presence of calcium this peptide forms a symmetric two-site homodimeric calcium-binding domain (Shaw et al., 1990). The solution structure of this dimer was determined from the measurement of 470 NOEs from a 75-ms NOESY data set. For the dimer structure determination, the constraint list included 868 distance restraints, 44 phi angles, and 24 chi 1 and 2 chi 2 angles. Seven structures were calculated by restrained molecular dynamics using a procedure in which intramonomer distances were used first and then all distances, intra- and intermonomer, were input during further dynamics. The structures exhibited a fold very similar to the C-terminal domain of troponin-C comprised of a pair of helix-loop-helix calcium-binding sites. The rms deviation of these structures for backbone atoms between residues 97-122 and 97'-122' for the dimer was 0.82 A. The dimer structure was also calculated to be more symmetric than sites III and IV in troponin-C.

Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

Biochemistry. 1992; 31: 7970-6

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A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.

Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on p11, a member of the S100 protein family.

J Biol Chem. 1992; 267: 14175-82

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p11, a member of the S100 protein family, forms a stable heterotetrameric complex with annexin II. The p11-binding site of annexin II resides in the N-terminal 14 residues, which form an amphiphatic alpha-helix with the hydrophobic face representing the contact site for p11 (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7, 2435-2442). We show that a corresponding peptide can be used to purify recombinant p11 by affinity chromatography. To map the annexin II-binding site on p11, we have produced progressively truncated p11 derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin II-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin II. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p11 for annexin II, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin II and p11 are involved in complex formation.

Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.

Biochemistry. 1992; 31: 9665-72

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The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)

Unlike human, rat and mouse calcyclin, purified rabbit calcyclin did not form a dimer on Tricine SDS-PAGE under non-reduced conditions. Based on the internal peptide sequence of rabbit calcylin, we isolated and sequenced a cDNA clone encoding calcyclin. The sequence of this clone (pCalC) is 629 bp long and codes 90 amino acid residues of a protein with a molecular mass of 10,153 Da. By Northern blot analysis, a major band of 0.9 kbp and a minor band of 2.6 kbp were detected in the lung. The recombinant calcyclin mutated serine at the third position to cysteine was expressed in E. coli and made dimer formation under non-reduced conditions on SDS-PAGE. Whether or not this type of mutation which prevents dimer formation of calcyclin plays a physiological role in the rabbit lung is the subject of an ongoing study.

A calcyclin-associated protein is a newly identified member of the Ca2+/phospholipid-binding proteins, annexin family.

J Biol Chem. 1992; 267: 8919-24

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A calcyclin-associated protein with an apparent molecular weight of 50,000 (CAP-50) was purified from rabbit lung. The procedure included ammonium sulfate precipitation, anion and cation ion-exchange, and calcyclin affinity chromatographies. Interestingly, partial amino acid sequences of lysyl-endpeptidase-digested fragments indicated that CAP-50 was a member of the Ca2+/phospholipid-binding proteins, the annexin family. The sequence of a proteolytic peptide with Staphylococcus aureus V8 protease on NH2-terminal region is not homologous with any other annexin family proteins. Phospholipid binding studies showed that CAP-50 bound to phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid-containing vesicles, in a Ca(2+)-dependent manner. In the presence of Ca2+/calcyclin, CAP-50 formed a complex with calcyclin and bound to the PS-containing vesicles. The apparent Kd value of calcyclin for CAP-50 was calculated to be 1.61 x 10(-6) M. Zero-length cross-linking studies indicated that 1 mol of CAP-50 bound to an equimolar unit of calcyclin. CAP-50 inhibited the phospholipase A2 activity, dose-dependently (IC50 = 0.2 microM), however, calcyclin did not alter the inhibitory effect. With the 125I-calcyclin gel overlay method, calcyclin bound tightly to CAP-50 in a Ca(2+)-dependent manner after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that rabbit lung CAP-50 is a newly identified member of the annexin family. Ca2+/calcyclin apparently regulates the function of CAP-50 on cytosolic face of the plasma membrane.

The 23-kilodalton protein, a substrate of protein kinase C, in bovine neutrophil cytosol is a member of the S100 family.

Biochemistry. 1992; 31: 5898-905

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A bovine neutrophil protein termed p23 because of an apparent molecular mass of 23 kDa in SDS-PAGE is present in large amounts both in a soluble form in the cytosolic fraction of bovine neutrophil homogenates and associated to the cytoskeleton. P23 is accompanied during the first steps of the purification procedure by a smaller size protein termed p7 on the basis of a rate of migration in SDS-PAGE corresponding to a 7-kDa protein [Stasia, M. J., Dianoux, A. C., & Vignais, P. V. (1989) Biochemistry 28, 9659-9667]. The two proteins, p23 and p7, have been purified to homogeneity by an improved procedure consisting of two chromatographic steps. The electrospray mass spectrometry technique applied to p23 and p7 indicated molecular masses close to 17 and 10 kDa, respectively, significantly different from the masses derived by SDS-PAGE. Bovine neutrophil p23 and p7 presented large primary structure homologies with two human proteins, MRP14 and MRP8, which are expressed in large amounts in macrophages under conditions of chronic inflammation. In addition, p23 and p7 cross-reacted with monoclonal antibodies specific of MRP14 and MRP8. Bovine p23 and p7 bound Ca2+, and their amino acid sequences contained two Ca(2+)-binding domains per protein, largely identical to those of human MRP14 and MRP8. Bovine p23 and p7 associated together to form a heterodimeric complex, which largely escaped attack by trypsin, whereas the isolated p23 and p7 components were readily digested. These features are typical of Ca(2+)-binding proteins belonging to the S100 family.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of recoverin-like Ca(2+)-binding protein from rat brain.

Biochem Biophys Res Commun. 1992; 183: 245-51

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Rat brain was found, by immunoblot analysis, to have a protein of Mr 23,000 (P23k) that was clearly different from recoverin and was labeled with an antiserum raised against the NH2-terminus of recoverin. P23k could not be detected by an antiserum raised against the COOH-terminus of recoverin. Blots with 45Ca demonstrated that P23k bound Ca2+. This calciprotein was further purified by Ca(2+)-dependent hydrophobic interaction and ion-exchange chromatography. In SDS polyacrylamide gel electrophoresis, P23k had an apparent Mr of 21,000 in the presence of 10 microM Ca2+ and 23,000 in the absence of Ca2+ (0.1 mM EGTA). The isoelectric point of P23k was 5.6. Ca(2+)-binding analysis indicated that P23k bound 2 moles of Ca2+ per mole of protein and had two binding sites with dissociation constants of 13 microM and 0.2 microM. Purified P23k bound to the crude membrane fractions from the cerebellum, cerebrum and retina in a Ca(2+)-dependent manner. Partial amino acid sequence analysis of proteolytic fragments of P23k revealed the sequence homology between P23k and recoverin. These results suggested that P23k may act as a Ca(2+)-sensitive regulator by forming a complex with its target on the membrane.

Calcium ions exert their effects in part via interactions with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has over 200 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. Some members, like parvalbumin, calbindin, and calretinin, proved to be useful neuronal markers for a variety of functional brain systems and their circuitries. Their major role is assumed to be buffering, transport of Ca2+, and regulation of various enzyme systems. Since cellular degeneration is accompanied by impaired Ca2+ homeostasis, a protective role for Ca(2+)-binding proteins in certain neuron populations has been postulated. Another protein family are the annexins, members of which interact with phospholipids and cellular membranes in a calcium-dependent manner. In some cases members of the annexin family were even found to interact with EF-hand proteins. Certain annexins have been suggested to be involved in anti-inflammatory response, inhibition of blood coagulation, membrane trafficking or cytoskeletal organization, but several of these functions have been questioned recently. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

Three peptides containing the putative Ca2+ binding loops, I, II and III, respectively, of a photoprotein, aequorin, from jellyfish Aequorea victoria were synthesized by a solid-phase procedure. The peptides bound Ca2+ with dissociation constants of 10(-3) to 10(-4) M, providing evidence for the assumption that Ca2+ binding loops are actually responsible for the binding of Ca2+. When the highly conserved 6th glycine residue in the 12-residue loops was replaced by arginine, no large effect was observed on Ca2+ binding. Exposure to a hydrophobic environment and the binding of Ca2+ brought about conformational changes to the peptides.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the Forster equation in approximating distances in these systems.

Previously we reported the amino acid sequences of 4 well-defined sacroplasmic, high-affinity Ca(2+)-binding proteins in the protochordate amphioxus, Branchiostoma lanceolatum [1]. Here we report on the complete amino acid sequence determination of 3 additional minor isoforms. The seven isoforms differ from each other in 9 positions of a contiguous 17-residue-long segment (positions 20-36) and can be classified in a alpha (ASCP I, III and IV) and a beta lineage (ASCP II, V, VI and VII).

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.

Using Ca(2+)-dependent affinity chromatography on a synthetic compound (W-7)-coupled Sepharose column, three distinct Ca(2+)-binding proteins have been identified in human platelets. The molecular mass of these three distinct proteins was estimated to be 10, 10.5, 17 kDa, respectively, by polyacrylamide gel electrophoresis in the presence of SDS. The partial amino acid sequence revealed these proteins have EF-hand structures and high homology to the predicted proteins, calcyclin, calvasculin, and calmodulin. Calcyclin and calvasculin have been considered as probably having roles in the control of cell proliferation, but the existence of these two proteins in platelets suggests that they have other intracellular functions related to the Ca(2+)-signal transduction system.

The protein hyalin, a major component of the sea urchin extraembryonic hyaline layer, was previously shown to undergo a Ca(2+)-induced self-association into large aggregates (gelation). This reaction represented a major step in assembly of the layer. In the experiments reported here, digestion with trypsin resulted in a rapid dissociation of hyalin into a mixture of peptides which retained the capacity to bind Ca2+. However, unlike intact hyalin, none of these peptides associated into large aggregates (gelation) in the presence of Ca2+, Mg2+, and NaCl. Loss of the ability to undergo gelation was not accompanied by any significant change in the content of acidic plus amide amino acid residues. Decreasing the pH to 5.6 resulted in a loss of 25% of hyalin's Ca(2+)-binding capacity but had no effect on the ability of the protein to undergo gelation. Peptide fragments were only partially effective at inhibiting hyalin gelation. Clearly, not all the Ca(2+)-binding sites were required for hyalin gelation and Ca2+ binding alone was insufficient to drive this reaction. In addition, hyalin appeared to possess two classes of protein-protein interaction domains, one of which was essential for gelation.

The Ca(2+)-and Mg(2+)-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, Gly108, Tyr109, Ile110, His125, Gly144, Ile146, Phe102 or Phe151, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[TnC] = 1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1 x 10(5) M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9 x 10(4) M-1, which is one-tenth of that of Ca4TnC.(ABSTRACT TRUNCATED AT 250 WORDS)

In a structure of recombinant bovine calbindin D9k, determined crystallographically to 1.6 A resolution, a proline in mixed, approximately equally populated, cis and trans conformation is observed. Isomers of this kind have not been reported in structure determinations of calbindin D9k to 2.3 A resolution or in any other crystallographically determined protein structure. The cis-trans isomerization occurs at the peptide bond between Gly42 and Pro43, which is in agreement with results from two-dimensional 1H nuclear magnetic resonance spectroscopy experiments on solutions of calbindin D9k. Alternative backbone stretches have been modeled and refined by stereochemical restrained least-squares refinement for the segment Lys41 to Pro43. The final R-value was 0.188. The structural perturbations accompanying the cis-trans isomerization are found to be very localized. The largest positional differences are observed at residue Gly42, in which the alternative positions of the oxygen atom are 3.6 A apart.

Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage.

Blood. 1992; 79: 1907-15

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MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.

Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.

Nereis sarcoplasmic calcium-binding protein contains three functional EF-hand sites which bind Ca2+ or Mg2+ competitively. Here it was confirmed over a large range of [Mg2+] that the positive cooperativity in binding of Ca2+ (nH = 2) is the result of allostery in Mg2+ dissociation. At pH 7.5, Ca2+ or Mg2+ binding provokes the release of 1.4 mol proton/mol protein, whereas no protons are released during Ca(2+)-Mg2+ exchange. The enthalpy change as a function of Ca2+ binding yields a two-step curve with an inflection point at 1 mol Ca2+/mol protein and a maximum of -66 kJ/mol at 3 mol Ca2+/mol protein. Binding of three Mg2+ ions is cooperative (nH = 1.8) with a maximal enthalpy change of -15.1 kJ/mol protein. Difference spectroscopy led to the conclusion that, in the metal-free protein, the structure around the aromatic residues is well organized, but that Tyr and Trp residues are still solvent-exposed. Upon Ca2+ binding Tyr and Trp spectra are blue-shifted, but some Trp residues are confined to a positively charged pocket. Examination of the Ca(2+)-saturated three-dimensional crystal structure confirmed that Trp4 and Trp57 are located in such pockets or clefts, close to the surface. During the allosteric T----R transition, promoted by binding of the first Mg2+, the Trp residues move to a hydrophobic environment. For both Ca2+ and Mg2+, the enthalpy change and the conformational change in the environment of the aromatic residues is much more pronounced in the first, than in the subsequent two binding steps. In this respect, the latter seem to be equivalent.

15N has been uniformly incorporated into the EF-hand Ca(2+)-binding protein calbindin D9k so that heteronuclear experiments can be used to further characterize the structure and dynamics of the apo, (Cd2+)1 and (Ca2+)2 states of the protein. The 15N NMR resonances were assigned by 2D 15N-resolved 1H experiments, which also allowed the identification of a number of sequential and medium-range 1H-1H contacts that are obscured by chemical shift degeneracy in homonuclear experiments. The 15N chemical shifts are analyzed with respect to correlations with protein secondary structure. In addition, the changes in 15N chemical shift found for the apo----(Cd2+)1----(Ca2+)2 binding sequence confirm that the effects on the protein are mainly associated with chelation of the first ion.

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.

1. Calcium (Ca)-binding proteins of neuronal ganglia and of single, identified neurons of the marine mollusk, Aplysia californica, were investigated. Using transblot/45Ca overlays two proteins, at Mr 45,000 and Mr 23,000, with a high Ca-binding ability were found. 2. Western blot analysis revealed that the protein at Mr 45,000 could be separated by 2D-PAGE into proteins with Mr 40,000 and Mr 43,000. The protein at Mr 40,000 immunocross-reacted with antisera directed against parvalbumin and rat calbindin D-28K, indicating a novel Ca-binding protein sharing common antigenic determinants for both proteins. 3. The protein at Mr 23,000 could be separated into a group of proteins with Mr 13,000-20,000 which showed a high degree of similarity to sarcoplasmatic calcium-binding proteins (SCP). 4. We further investigated the protein pattern of single, identified neurons of different electrical activity (bursting, beating, and silent) by 2D-PAGE. Major differences were found in the range of low Mr and low pI, where Ca-binding proteins are generally located. A protein at high concentrations characteristic for silent cells migrated at a position similar to crayfish SCP. 5. The results show that various Ca-binding proteins are characteristic for neurons in the Aplysia nervous system and support the idea that they may effect the electrical behavior of nerve cells.

Molecular cloning and expression of the cDNA coding for a new member of the S100 protein family from porcine cardiac muscle.

FEBS Lett. 1991; 295: 93-6

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We isolated a new calcium-binding protein from porcine cardiac muscle by calcium-dependent hydrophobic and dye-affinity chromatography. It showed an apparent molecular weight of 11,000 on SDS-PAGE. Amino acid sequence determination revealed that the protein contained two calcium-binding domains of the EF-hand motif. The cDNA gene coding for this protein was cloned from the porcine lung cDNA library. Sequence analysis of the cloned cDNA showed that the protein was composed of 99 amino acid residues and its molecular weight was estimated to be 11,179. Immunological and functional characterization showed that the recombinant S100C protein expressed in Escherichia coli was identical to the natural protein. Homologies to calpactin light chain, S100 alpha and beta protein were 41.1%, 40.9% and 37.5%, respectively. The protein was expressed at high levels in lung and kidney, and low levels in liver and brain. The tissue distribution was apparently different from those of the other S100 protein family. These results indicate that this protein represents a new member of the S100 protein family, and thus we refer to it as S100C protein.

Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy.

Biochemistry. 1991; 30: 4323-33

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The structure of a 39 amino acid proteolytic fragment of rabbit skeletal troponin C containing the fourth Ca(2+)-binding site has been determined by an approach involving nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Hydrodynamic and NMR evidence establishes unambiguously that the fragment forms a stable dimer in solution in the presence of excess Ca2+. The calculation of the dimeric structure is based on a total of 1056 experimental restraints comprising 422 interproton distances, 35 phi, 28 psi, and 28 chi 1 torsion angle restraints within each subunit, 30 intermonomer distance restraints, and 6 Ca2+ restraints per subunit. A total of 48 final structures were calculated having an rms deviation about the mean atomic backbone coordinate positions of 1.0 A for residues Asp128-Glu156. The solution structure consists of a dimer of helix-loop-helix motifs related by a 2-fold axis of symmetry. The overall architecture of the dimer is very similar to the C-terminal domain in the crystal structure of chicken skeletal troponin C.

Intracellular calcium transients play a major role in the control of cellular contraction and act through binding to target proteins and inducing subsequent conformational changes and activation of enzymes. Abnormalities of intracellular calcium handling are involved in the pathophysiology of essential hypertension and cardiac hypertrophy. In this study we report on the isolation, purification and calcium binding of a 33 kDa protein from human platelets and of a 38 kDa protein from cardiac tissue, both of which are identified as tropomyosin. The calcium binding properties of these human tropomyosin isoforms indicate a putative role for these proteins in the fine tuning of the cellular contraction. Elevated tropomyosin level is demonstrated in platelets from untreated essential hypertensive patients with left ventricular hypertrophy (tropomyosin/actin: 45.1 +/- 3.5, n = 12) relative to essential hypertensive patients without cardiac hypertrophy (tropomyosin/actin: 33.8 +/- 2.3). These findings suggest an association between the enhanced expression of tropomyosin in platelets and the development of cardiac hypertrophy which may relate to the cellular calcium overload of this disease.

The effects of secretagogue(s) from mouse decidual tissue on the release of mouse placental lactogen-II (mPL-II) were studied. Decidual tissue was obtained from 10- and 11-day-pregnant mice. The tissue was homogenized, extracted, and the tissue extract was made 50% saturated with ammonium sulfate. Both the precipitate and supernatant were tested for their ability to stimulate mPL-II release from cultured trophoblasts. The supernatant contained an activity to stimulate the release of mPL-II. This activity was further purified using column chromatography. The purification resulted in isolation of a protein with a mol wt of 20 K as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 6 K under reducing conditions. Further characterization of this protein showed that it binds calcium and has an amino acid sequence that is highly homologous with calcyclin expressed in mouse embryonic fibroblast cells and with calcyclin from other species. This protein was designated mouse decidual calcyclin. Antiserum was raised against the purified decidual calcyclin for development of an RIA and for immunoblots. Western blots of various mouse tissue extracts and mouse serum from different physiological stages showed that the concentration of calcyclin was highest in decidual tissue. Detectable levels were found in extracts from trophoblast, lung, and stomach, but the concentrations in these tissues were about 100 times lower than in decidua. Decidual calcyclin was not detectable in mouse serum. Cultured decidual cells released calcyclin into the medium. On average, this release was about 7.8 ng/micrograms DNA.24 h. The rate of release did not change significantly during 4 days of culture. The ratio of calcyclin in cells per calcyclin released during 24 h averaged 2.3 and did not change significantly during the culture period. The purified decidual calcyclin stimulated the release of mPL-II from cultured trophoblasts in a dose-dependent manner at concentrations from 0.01 to 1 microgram/ml. The maximum stimulation averaged about 1.5 times above control. It is concluded that decidual calcyclin may be of physiological importance for the regulation of mPL-II secretion.

High-resolution solution structure of reduced French bean plastocyanin and comparison with the crystal structure of poplar plastocyanin.

J Mol Biol. 1991; 221: 533-55

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The three-dimensional solution structure of reduced (CuI) plastocyanin from French bean leaves has been determined by distance geometry and restrained molecular dynamics methods using constraints obtained from 1H n.m.r. (nuclear magnetic resonance) spectroscopy. A total of 1244 experimental constraints were used, including 1120 distance constraints, 103 dihedral angle constraints and 21 hydrogen bond constraints. Stereospecific assignments were made for 26 methylene groups and the methyls of 11 valines. Additional constraints on copper co-ordination were included in the restrained dynamics calculations. The structures are well defined with average atomic root-mean-square deviations from the mean of 0.45 A for all backbone heavy atoms and 1.08 A for side-chain heavy atoms. French bean plastocyanin adopts a beta-sandwich structure in solution that is similar to the X-ray structure of reduced poplar plastocyanin; the average atomic root-mean-square difference between 16 n.m.r. structures and the X-ray structure is 0.76 A for all backbone heavy atoms. The conformations of the side-chains that constitute the hydrophobic core of French bean plastocyanin are very well defined. Of 47 conserved residues that populate a single chi 1 angle in solution, 43 have the same rotamer in the X-ray structure. Many surface side-chains adopt highly preferred conformations in solution, although the 3J alpha beta coupling constants often indicate some degree of conformational averaging. Some surface side-chains are disordered in both the solution and crystal structures of plastocyanin. There is a striking correlation between measures of side-chain disorder in solution and side-chain temperature factors in the X-ray structure. Side-chains that form a distinctive acidic surface region, believed to be important in binding other electron transfer proteins, appear to be disordered. Fifty backbone amide protons form hydrogen bonds to carbonyls in more than 60% of the n.m.r. structures; 45 of these amide protons exchange slowly with solvent deuterons. Ten hydrogen bonds are formed between side-chain and backbone atoms, eight of which are correlated with decreased proton exchange. Of the 60 hydrogen bonds formed in French bean plastocyanin, 56 occur in the X-ray structure of the poplar protein; two of the missing hydrogen bonds are absent as a result of mutations. It appears that molecular dynamics refinement of highly constrained n.m.r. structures allows accurate prediction of the pattern of hydrogen bonding.

Calcium binding to proteins containing the 'EF-hand' structural motif regulates a variety of biochemical processes including muscle contraction. Techniques such as protein crystallography, site-directed mutagenesis and domain transplantation experiments are being used to unravel the conformational changes induced by calcium binding.

The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.

The primary structure of bovine retinal calcium binding protein P26 has been determined by parallel analysis of protein and corresponding cDNA. This protein is identical to recovering and shares 59% homology with visinin, a cone specific calcium binding protein from chicken retina. Preliminary data are presented on expression of P26 as a fusion protein in E. coli.

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy.

J Cell Biol. 1991; 112: 981-9

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Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity-purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure.

Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution.

J Biol Chem. 1991; 266: 21375-80

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The crystal structure of calmodulin (Mr 16,700, 148 residues) from Drosophila melanogaster as expressed in a bacterial system has been determined and refined at 2.2-A resolution. Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for the 5,239 reflections (F greater than or equal to 2 sigma (F)) within the 10.0-2.2-A resolution range. The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mean square deviations from standard values of 0.018 A for bond lengths and 0.043 A for angle distances. The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calcium-binding domains. The "dumb-bell" shaped molecule contains seven alpha-helices and four "EF hand" calcium-binding sites. Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures. Superimposition of the structures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms. The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms. There are also differences in the amino terminus, the bend of the central alpha-helix, and the orientations of some of the side chains.

Mutation of the pseudo-EF-hand of calbindin D9k into a normal EF-hand. Biophysical studies.

Eur J Biochem. 1991; 202: 1283-90

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The two Ca(2+)-binding sites in calbindin D9k, a protein belonging to the calmodulin superfamily of intracellular proteins, have slightly different structure. The C-terminal site (amino acids 54-65) is a normal EF-hand as in the other proteins of the calmodulin superfamily, while the N-terminal site (amino acids 14-27) contains two additional amino acids, one of which is a proline. We have constructed and studied five mutants of calbindin D9k modified in the N-terminal site. In normal EF-hand structures the first amino acid to coordinate calcium is invariantly an Asp. For this reason Ala15, is exchanged by an Asp in all mutants and the mutants also contain various other changes in this site. The mutants have been characterized by 43Ca, 113Cd and 1H NMR and by the determination of the calcium binding constants using absorption chelators. In two of the mutants (one where Ala14 is deleted, Ala15 is replaced by Asp and Pro20 is replaced by Gly, the other where, in addition, Asn21 is deleted), we find that the structure has changed considerably compared to the wild-type calbindin. The NMR results indicate that the calcium coordination has changed to mainly side-chain carboxyls, from being octahedrally coordinated by mainly back-bone carbonyls, and/or that the coordination number has decreased. The N-terminal site has thus been turned into a normal EF-hand, in which the calcium ion is coordinated by side-chain carboxyls. Furthermore, the calcium binding constants of these two mutant proteins are almost as high as in the wild-type calbindin D9k. That is, the extensive alterations in the N-terminal site have not disrupted the calcium binding ability of the proteins.

Annexins are a family of water-soluble proteins that bind to membranes in a calcium-dependent manner. Some members have been shown to exhibit voltage-dependent calcium channel activity, a property characteristic of integral membrane proteins. The structures of human annexin V in crystals obtained from aqueous solution and in two-dimensional crystals when bound to phospholipid layers have been determined by X-ray and electron crystallography, respectively. They are compared here. Both structures show close correspondence, suggesting that annexins attach to phospholipid membranes without substantial structural change. These observations, together with biochemical data, lead to the conclusion that annexin V interacts with phospholipid membranes with its convex face. We propose that binding is mediated by direct interaction between the phosphoryl headgroups and the calcium bound to polypeptide loops protruding from the convex face. The membrane area covered by annexin may thus become disordered and permeable allowing calcium flux through the membrane and the central channel-like structure found in annexin molecules.

Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments.

J Mol Biol. 1991; 220: 1017-39

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The crystal structure of the Ca-loaded form of pike 4.10 parvalbumin (minor component from pike muscle belonging to the beta phylogenetic series), with both its primary sites CD and EF occupied by Ca2+ ions and its third site occupied by an ammonium ion, as previously determined at 1.93 A resolution, has now been refined to a resolution of 1.65 A. The crystallization of this parvalbumin in different ionic environments has allowed three novel non-isomorphous crystalline forms to be obtained: (1) a first form, crystallized in the presence of a mixture of ammonium sulphate and manganese sulphate, for which all the cation binding sites in the protein are occupied by Mn2+; (2) a second form crystallized in the presence of MgSO4 as the precipitating agent, only differs from the Ca/NH4 form by the occupation of the third site by Mg2+, whereas the primary sites remain occupied by Ca2+; (3) a third form, also crystallized in the presence of MgSO4, corresponds to a well-defined molecular species with both the primary EF site and the third site occupied by Mg2+, whereas the primary CD site remains occupied by CA2+. The corresponding molecular structures reported here have been determined at resolutions between 1.8 and 2.4 A. The comparison of the different crystal structures allows the structural modifications accompanying the substitution of the primary sites by cations differing significantly in their ionic radii (Ca2+, Mn2+, Mg2+) to be investigated in detail, and it also leads to a precise description of the third site in a typical beta parvalbumin. The substitution Ca2+ by Mg2+ within the primary site EF is characterized by a "contraction" of the co-ordination sphere, with a decrease of the mean oxygen-metal distance by a value of 0.25 A and a decrease of the co-ordination number from 7 to 6, as a consequence of the loss of a bidentate ligand (Glu101), which becomes a monodentate one. Such an adaptation of the co-ordination sphere around a cation of smaller size involves, among others, the transformation of the Glu101 side-chain from the stable gauche(+) form to the less stable gauche(-) form. The third site is clearly described as a satellite of the CD primary site, since both sites possess common protein ligands, such as Asp53 and Glu59. Furthermore, Asp61 appears as a specific ligand of the third site in the different environments investigated in this work. We finally discuss the relevance of the third site to parvalbumin phylogeny.

Identification and immunolocalization of calreticulin in pancreatic cells: no evidence for "calciosomes".

Exp Cell Res. 1991; 197: 91-9

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In the present study, we have shown that calreticulin is a major Ca(2+)-sequestering protein in pancreatic microsomes. This protein is a peripheral membrane protein and could be extracted from the microsomal membrane with carbonate buffer at pH 11.4. Calreticulin was identified in the membrane fractions by immunoblotting with a specific antibody, by a 45Ca2+ overlay technique, and by NH2-terminal amino acid analysis of the purified protein. Immunocytochemical localization of calreticulin in pancreatic acinar cells and pancreatic fibroblasts showed that the protein is localized to the ER membranes in these cells. We were unable to detect calsequestrin or any calsequestrin-like proteins in the pancreas and found no evidence for the existence of large numbers of specialized, calreticulin-containing vesicles which could be an equivalent of the calsequestrin-containing calciosomes previously reported in this tissue. Purified pancreatic calreticulin binds Ca2+ with both a low and a high capacity (approximately 1 mol of Ca2+/mol of protein and approximately 20-23 mol of Ca2+/mol of protein). The concentrations of Ca2+ required for half-maximal saturation of the low and high capacity sites were approximately 4-6 microM and approximately 1.5 mM, respectively. We conclude that calreticulin, which is confined to the lumen of the ER, plays a major role in Ca2+ storage in pancreatic cells.

A synthetic 27-residue peptide corresponding to exon 1B of the endogenous inhibitor calpastatin contains a well-conserved region and has an ability to inhibit the cysteine endopeptidase calpain specifically. We examined the solution structure of this peptide in DMSO-d6 by two-dimensional 1H NMR spectroscopy. Although regular secondary structures such as alpha-helix and beta-sheet were not found, the region from Ile18 to Arg23 formed a well-defined structure with a type I beta-turn. This region coincided well with the highly conserved region of calpastatin. The result strongly suggests that this turn structure is essential for the inhibitory activity of calpastatin.

Selective secretion of annexin 1, a protein without a signal sequence, by the human prostate gland.

J Biol Chem. 1991; 266: 2499-507

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Annexins are primarily intracellular proteins as would be predicted from their lack of hydrophobic signal sequences. However, we now report that the human prostate gland selectively secretes high concentrations of annexin 1 (also called lipocortin 1 and p35) and a proteolytic cleavage product, des1-29-annexin 1, into seminal plasma. Secreted annexin 1 had a blocked amino terminus and was structurally indistinguishable from intracellular annexin 1. Although annexin 1 and the structurally related protein, annexin 4, co-localized to many of the same cells of the ductal epithelium of the prostate, annexin 4 was not secreted. Thus, the secretion of annexin 1 appears to involve a highly selective mechanism that does not involve targeting to the endoplasmic reticulum by a hydrophobic signal sequence.

Quantitating and engineering the ion specificity of an EF-hand-like Ca2+ binding.

Biochemistry. 1991; 30: 8690-7

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The Escherichia coli D-galactose and D-glucose receptor, an aqueous periplasmic receptor that triggers sugar sensing and transport, possesses a single Ca2+ binding site similar in structure and specificity to the EF-hand class of sites found in eukaryotic Ca2+ signaling proteins including calmodulin and its homologues. A universal feature of these sites is the use of a pentagonal bipyramidal array of seven oxygens to coordinate bound Ca2+. Here we investigate the mechanisms used by this coordinating array to control ion specificity. To vary the cavity size and charge of the array, we have replaced axial glutamine 142 in the prokaryotic site with asparagine, glutamate, and aspartate. The ion selectivities of the resulting engineered sites have been quantitated by measuring dissociation constants for a series of spherical metal ions, differing in increments of radius and charge, from groups Ia, IIa, and IIIa and the lanthanides. Dramatic specificity changes are observed: sites containing an engineered smaller side chain (Asn or Asp) bind the largest cations up to 50-fold more tightly than the native site; and sites containing an engineered negative side chain (Glu or Asp) exhibit preferences for trivalent over divalent cations up to 1900-fold higher than the native site. The results indicate that the cavity size and negative charge of the coordination array play key roles in selective Ca2+ binding and that the array can be engineered to preferentially bind other cations.

Despite striking sequence homology with rat parvalbumin, oncomodulin exhibits much lower affinity for Ca2+ ion. We are attempting to identify the structural basis for this difference by systematically substituting the parvalbumin residue for the oncomodulin residue at points of nonidentity. In this paper, we examine two mutations in the helical segments flanking the CD ion-binding loop. Replacement of Asp-45 in the C helix by lysine, to produce D45K, reduces the dissociation constant for Ca2+ at the CD site from 0.81 to 0.53 microM. Replacement of Lys-69 in the D helix by glycine, to afford K69G, similarly reduces KCa to 0.59 microM. Both mutations perturb the Eu3+ 7Fo----5Do spectral parameters. We also examine the consequences of simultaneous mutations involving positions 57, 59, 60, and 69. Ca(2+)-binding assays and Eu3+ luminescence measurements indicate that there is a conformational interaction between residues 57 and 69 and that this interaction is modulated by residues 59 and 60. When the mutations at positions 57, 59, 60, and 69 are combined, the resulting variant exhibits a KCa value for the CD site of 0.25 microM, reflecting a 3-fold increase in affinity relative to the wild-type protein. Moreover, the pK alpha governing the interconversion of low and high pH forms of the Eu3+ 7Fo----5Do spectrum is increased to 8.1, very close to the value of 8.25 determined previously for rat parvalbumin. In this paper, we also complete our survey of single mutations in the CD loop by examining L58I. Replacement of Leu-58 by isoleucine reduces the affinity of the CD site for Ca2+, raising KCa to 2.2 microM. Finally, we revise our previous estimate of the KCa value for Y57F downward, from 0.80 to 0.64 microM. The earlier result is believed to have been inflated by heterogeneity in the preparation, a consequence of proteolysis.

In a large number of intracellular regulatory proteins of the calmodulin superfamily a pair of closely interacting helix-loop-helix Ca2+ binding sites ('EF hands') constitute the functional unit--an arrangement that enables cooperative binding. We have recently made detailed experimental studies of the binding of Ca2+ ions to calmodulin, its tryptic fragments TR1C and TR2C (which each constitute a globular domain of a pair of EF hands) and calbindin D9k. Macroscopic Ca2+ binding constants have been obtained over a range of ionic strengths (0 to 0.15 M KCl). For calmodulin the measurements indicate that the two separate globular domains TR1C and TR2C retain the Ca2+ binding properties they have in the intact molecule, with positive cooperativity within each domain. The absolute value of the free energy of interaction between the two sites in each domain, a measure of the cooperativity, increases with ionic strength and is greater than or equal to 10 kJ mol-1 at 0.15 M KCl. Two-dimensional 1H NMR studies show that the addition of KCl does not alter the conformation of the protein. In the case of calbindin D9k several categories of mutants have been studied. One group encompasses the effect of protein surface charges 5 to 15 A from the Ca2+ binding sites. Two-dimensional 1H NMR shows that neither the addition of KCl, nor mutations that neutralize the surface charges, change the protein conformation. Although the global structure of calbindin D9k is largely unchanged upon binding of calcium, the structure with only one cation bound is more similar to the (Ca2+)2 form. Interestingly, the dynamical properties of the Ca(2+)-free and the (Ca2+)2-forms of calbindin differ greatly. For example, the rate of NH/ND exchange of the Ca(2+)-free form is on average 200 times faster than that of the (Ca2+)2-form. The results obtained so far point to a non-negligible entropic contribution to the observed cooperativity of Ca2+ binding.

Characterization of a Ca(2+)-binding site in human annexin II by site-directed mutagenesis.

J Biol Chem. 1991; 266: 14732-9

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Annexin II, a major cytoplasmic substrate of the src tyrosine kinase, is a member of the annexin family of Ca2+/phospholipid-binding proteins. It is composed of a short N-terminal tail (30 residues) followed by four so-called annexin repeats (each 70-80 residues in length) which share sequence homologies and are thought to form (a) new type(s) of Ca(2+)-binding site(s). We have produced wild-type and site specifically mutated annexin II molecules to compare their structure and biochemistry. The recombinant wild-type annexin II displays biochemical and spectroscopical properties resembling those of the authentic protein purified from mammalian cells. In particular, it shows the Ca(2+)-induced blue shift in fluorescence emission which is typical for this annexin. Replacement of the single tryptophan in annexin II (Trp-212) by a phenylalanine abolishes the fluorescence signal and allows the unambiguous assignment of the Ca(2+)-sensitive spectroscopic properties to Trp-212. This residue is located in the third annexin repeat in a highly conserved stretch of 17 amino acids which are also found in the other repeats and known as the endonexin fold. To study the precise architecture of the Ca2+ site which must reside in close proximity to Trp-212, we changed several residues of the endonexin fold in repeat 3 by site-directed mutagenesis. An analysis of these mutants by fluorescence spectroscopy and Ca(2+)-dependent phospholipid binding reveals that Gly-206 and Thr-207 seem indispensible for a correct folding of this Ca(2+)-binding site.

Genomic structure of Chlamydomonas caltractin. Evidence for intron insertion suggests a probable genealogy for the EF-hand superfamily of proteins.

J Mol Biol. 1991; 221: 175-91

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A clone containing the gene locus for Chlamydomonas caltractin, a 20,000 Mr calcium-binding protein that is a member of the EF-hand superfamily of calcium-modulated proteins, was isolated and the structural organization of the gene was determined. The intron-exon organization was resolved by direct comparison of the genomic sequence with a caltractin cDNA. The promoter region does not contain the typical TATA or CCAAT boxes, but the sequences at the splice junctions are similar to those of other eukaryotes. The positions of the six introns in the caltractin gene do not typically define unit structures, nor do they coincide with those in genes for other members of the EF-hand superfamily. An analysis of exon sequences at the splice junctions in the genes of this multigene family was undertaken; evidence was obtained that supports the hypothesis that introns arose at proto-splice sites. A probable evolutionary history for the EF-hand superfamily based on intron insertion is offered.

The molecular basis for the co-operativity in binding of calcium ions by bovine calbindin D9k has been addressed by carrying out a comparative analysis of the solution conformation and dynamics of the apo, half saturated and fully saturated species using two-dimensional 1H nuclear magnetic resonance spectroscopy. Since the half saturated calcium form of the protein is not significantly populated under equilibrium conditions due to the co-operativity in binding of calcium ions, the half saturated cadmium form of the protein has been substituted for the calcium form. To verify that cadmium forms of calbindin D9k represent viable models for the calcium-bound species, the fully saturated cadmium form has been prepared and compared to the calcium-saturated protein. Virtually complete 1H resonance assignments have been obtained for both the (Cd2+)1 and the (Cd2+)2 states. Secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone spin-spin coupling constants and slowly exchanging amide protons. Comparisons of the half saturated protein with the apo and calcium-saturated forms of calbindin D9k show that all three structures are highly similar. However, a change in the structural and dynamic properties of the protein does occur upon binding of the first ion; the half saturated form is found to be more similar to the calcium-saturated form than to the apo form. These results have important implications concerning the molecular basis for the co-operativity, and suggest that entropic effects associated with the protein dynamics play an important role.

In order to understand the mechanism of the various responses evoked by calcium in the cell, the identification and characterization of a number of calcium receptors were undertaken within the past two decades. Advances in amino acid sequence and protein three-dimensional structure led to the description of two families of calcium-binding proteins, the EF-hand homolog family and the annexin family. The EF-hand motif consists of two alpha helices, "E" and "F", joined by a Ca(2+)-binding loop. EF-hands have been identified in numerous Ca(2+)-binding proteins by similarity of amino acid sequence and confirmed in some crystal structures. Functional EF-hands seem always to occur in pairs. To date, the EF-hand homolog family contains more than 160 different Ca(2+)-modulated proteins which have a broad range of functions. Among them, are the calmodulin, the troponin C, the myosin regulatory light chain, the parvalbumin, the S-100 proteins and the calbindins 9- and 28 kDa. The most striking feature of the EF-hand family is the ability to modulate the activity of a number of enzymes. Several groups have identified proteins from various tissues that show calcium-dependent binding to membranes. These proteins, termed annexins have a molecular weight of 35- or 67 kDa. The amino acid sequences of the members of the annexin family show that each protein contains conserved internal repeats of about 70 amino acids each. The 35 kDa annexins contain four repeats, which show a high degree of homology with each other and with the repeat sequences of the other proteins. These repeats correspond to structural domains with a similar fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and synthesis of the pseudo-EF hand in calbindin D9K: effect of amino acid substitutions in the alpha-helical regions.

Proteins. 1991; 9: 12-22

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A series of 37-residue analogues of the pseudo-EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an alpha-helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two alpha-helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu-...Lys+ salt bridge or by changing the N-terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices. The mutants with the substitution of (Leu-30, Leu-32) to (Gln-30, Gln-32), (Gln-30, Ala-32), and (Ala-30,Ala-32) designed based on the strategy 1 do not show any affinity for calcium and have low alpha-helicity. The Leu-30 to Lys-30 mutant designed to form a salt bridge between the side chains of Glu-26 and Lys-30 has an apparent Kd for calcium of 6.8 mM. Kd of the N-terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the alpha-helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr-13, Leu-31 to Cys-13, Cys-31 and Tyr-13, Leu-31 to Cys-13, hCys-31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest alpha-helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix-loop-helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.

Calcium ions as biological regulators exert their effects in part via interaction with a wide variety of intracellular calcium-binding proteins. One class of these proteins shares a common calcium-binding motif, the EF-hand. A consensus amino acid sequence for this motif has aided the identification of new members of this family of EF-hand proteins, which now has about 170 members. A few of these proteins are present in all cells, whereas the vast majority are expressed in a tissue-specific fashion. The physiological function of a few of these proteins is known to be achieved via a calcium-dependent interaction with other proteins, thereby regulating their activity. The elucidation of the interactions and functions of the majority of these proteins remains a challenging task for the coming years.

The kinetics of calcium binding to tissue substrates in hypercalcaemia, induced by intravenous constant rate infusion of calcium gluconate were studied in 19 patients after renal transplantation and in 14 healthy volunteers. Langmuir, Scatchard and Hill analysis revealed the positive cooperative mechanism of calcium binding to tissue in all patients and in healthy volunteers. These results provide indirect evidence of binding of calcium to tissue proteins. The significant decrease of the values of specific buffer capacity of tissue proteins, pool of exchangeable calcium and also the effective Ka were observed in 37% of patients. These changes are proposed to play a role in the pathological calcification of vessels and soft tissues in patients after renal transplantation.

Characterization of a discontinuous epitope on annexin II by site-directed mutagenesis.

FEBS Lett. 1991; 285: 59-62

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Recombinant annexin II mutants were generated to identify amino acids involved in the formation of the discontinuous epitope of the monoclonal antibody H28. Analysis of the various mutant proteins by immunoblotting and enzyme-linked immunosorbent assay revealed that residues Lys27, Arg62, Glu65, and Arg67 are indispensable for H28 reactivity. Residues in equivalent positions are also in close proximity in the recently determined X-ray structure of annexin V, a different member of the same family of Ca2+/lipid-binding proteins. Thus annexins II and V show a similar three-dimensional folding in this region of the molecule. Consequently, the Ca2+ binding sites and the residues phosphorylated by pp60src (Tyr23) and protein kinase C (Ser25) most likely reside on opposite sides of the annexin II molecule.

Possible mechanism for the influence of weak magnetic fields on biological systems.

Bioelectromagnetics. 1991; 12: 71-5

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A physical mechanism is suggested for a resonant interaction of weak magnetic fields with biological systems. An ion inside a Ca(2+)-binding protein is approximated by a charged oscillator. A shift in the probability of ion transition between different vibrational energy levels occurs when a combination of static and alternating magnetic fields is applied. This in turn affects the interaction of the ion with the surrounding ligands. The effect reaches its maximum when the frequency of the alternating field is equal to the cyclotron frequency of this ion or to some of its harmonics or sub-harmonics. A resonant response of the biosystem to the magnetic field results. The proposed theory permits a quantitative explanation for the main characteristics of experimentally observed effects.

Interaction of troponin I and troponin C: 19F NMR studies of the binding of the inhibitory troponin I peptide to turkey skeletal troponin C.

Biochem Cell Biol. 1991; 69: 674-81

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We have used 19F nuclear magnetic resonance spectroscopy to study the interaction of the inhibitory region of troponin (TnI) with apo- and calcium(II)-saturated turkey skeletal troponin C (TnC), using the synthetic TnI analogue N alpha-acetyl[19FPhe106]TnI(104-115)amide. Dissociation constants of Kd = (3.7 +/- 3.1) x 10(-5) M for the apo interaction and Kd = (4.8 +/- 1.8) x 10(-5) M for the calcium(II)-saturated interaction were obtained using a 1:1 binding model of peptide to protein. The 19F NMR chemical shifts for the F-phenylalanine of the bound peptide are different from the apo- and calcium-saturated protein, indicating a different environment for the bound peptide. The possibility of 2:1 binding of the peptide to Ca(II)-saturated TnC was tested by calculating the fit of the experimental titration data to a series of theoretical binding curves in which the dissociation constants for the two hypothetical binding sites were varied. We obtained the best fit for 0.056 mM less than or equal to Kd1 less than or equal to 0.071 mM and 0.5 mM less than or equal to Kd2 less than or equal to 2.0 mM. These results allow the possibility of a second peptide binding site on calcium(II)-saturated TnC with an affinity 10- to 20-fold weaker than that of the first site.

Gd3+ vibronic side band spectroscopy. New optical probe of Ca2+ binding sites applied to biological macromolecules.

Biophys J. 1991; 59: 1040-9

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A new spectroscopic technique is presented for obtaining infraredlike spectra of the binding sites of Ca2+ and other metals in biological macromolecules. The technique, based on the Ca(2+)-like binding properties of Gd3+, utilizes vibronic side bands (VSB) that appear in Gd3+ fluorescence. In the fluorescence spectrum of Gd3+, the separation in photon frequency between a VSB and its electronic origin at approximately 32,150 cm-1 (approximately 311 nm) is a direct measure of the vibrational frequency of a ligand coordinated to Gd3+ ion. As a consequence, the VSB are uncomplicated by molecular vibrations distant from the Gd3+ binding site. The vibrational spectra resulting from the VSB of Gd3+ coordinated to a Ca2+ binding protein, a phospholipid, and DNA are presented.

Monte Carlo simulations are used to calculate the binding constant of two Ca2+ ions to the protein bovine calbindin D9k. The change in binding constant with respect to mutation of charged amino acids, presence of various electrolytes, protein concentration, solution pH, and competitive binding of monovalent ions is investigated. Each of these factors may have a large influence on the binding constant. The simulations are performed in a dielectric continuum model, the so-called primitive model of electrolyte theory, with a fixed protein structure and a uniform dielectric permittivity. The calculated binding constants are in excellent agreement with experimental data and describe changes in the binding constant over six orders of magnitude.

Gel filtration studies show that the thyroglobulin (Tg) molecule (dimer) binds from 18 to 50 Ca2+ ions. At pH 7.5 Tg binds 18 Ca2+ ions with a Kd of 1.3 x 10(-5) M, and 50 Ca2+ ions with a Kd of 5.5 x 10(-4) M. The binding of calcium to bovine thyroglobulin increases the absorption band of iodoamino acid residues at 315 nm. In the presence of Ca2+, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) is increased about 5-fold by Tg, with a shift in the fluorescence emission maximum from 505 to 490 nm. Thus, thyroglobulin possesses two classes of calcium binding sites with different affinities. The data reported indicate, also, that Ca2+ binding to Tg increases the hydrophobicity of the surface of the molecule.

Calcium-induced autolysis of bovine erythrocyte calpain I occurs in multiple stages. Initially, a 14 amino acid segment is cleaved from the N-terminus of the native 80 kDa catalytic subunit, yielding a 78 kDa form of the subunit. Then, an additional 12 amino acid segment is cleaved from the N-terminus, forming a 76 kDa subunit. The 76 kDa enzyme is the active form of the catalytic subunit that is able to proteolyze the 30 kDa regulatory subunit as well as exogenous substrates. While the initial autolytic step requires high calcium, the 76 kDa enzyme form is active in microM calcium and can cleave the amino termini of native 80 kDa and intermediate 78 kDa enzyme forms at low calcium. Both intramolecular and intermolecular proteolysis of the catalytic subunit appear to yield the same products.

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II.

Biochemistry. 1991; 30: 10236-45

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One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.

Probing the relationship between alpha-helix formation and calcium affinity in troponin C: 1H NMR studies of calcium binding to synthetic and variant site III helix-loop-helix peptides.

Biochemistry. 1991; 30: 8339-47

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Three 34-residue peptides corresponding to the high-affinity calcium-binding site III and two variant sequences from the muscle protein troponin C (TnC) were synthesized by solid-phase techniques. The two variant 34-residue peptides had amino acid modifications at either the coordinating positions or both the coordinating and noncoordinating positions, which corresponded to the residues found in the low-affinity calcium-binding site II of TnC. High-field 1H NMR spectroscopy was used to monitor calcium binding to each peptide to determine the effect these amino acid substitutions had on calcium affinity. The dissociation constant of the native site III peptide (SCIII) was 3 x 10(-6) M, smaller than that of the peptide incorporating the ligands from site II (LIIL), 8 x 10(-6) M, and that with the entire site II loop (LII), 3 x 10(-3) M, which bound calcium very weakly. These calcium dissociation constants demonstrate that very minor amino acid substitutions have a significant effect on the dissociation constant and give some insight into why the dissociation constants for site III and IV in TnC are 100-fold smaller than those for sites I and II. The results suggest that the differences in coordinating ligands between sites II and III have very little effect on Ca2+ affinity and that the noncoordinating residues in the site II loop are responsible for the low affinity of site II compared to the high affinity of site III in TnC.

Metal-induced changes in the fluorescence properties of tyrosine and tryptophan site-specific mutants of oncomodulin.

Biochemistry. 1991; 30: 7652-60

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Oncomodulin is a 108-residue, oncodevelopmental protein containing two calcium-binding sites identified as the CD- and EF-loops. The protein contains no tryptophan and only two tyrosine residues, one which is a calcium ligand in the CD-loop (Tyr-57) and one which lies in the flanking D-helix of this loop (Tyr-65). Site-specific mutagenesis was performed to yield five mutants, two with phenylalanine substituted for tyrosine in positions 57 and 65 and three with tryptophan substituted into position 57 in the CD-loop, position 65 in the D-helix, and position 96 in the EF-loop. The single Tyr-containing mutants demonstrated that position 57 was perturbed to a significantly greater extent than position 65 upon calcium binding. Although both tyrosine residues responded to decalcification, the fluorescence intensity changes were in opposite directions, with the more dominant Tyr-57 accounting for the majority of the intrinsic fluorescence observed in native oncomodulin. The substitution of tryptophan for each tyrosyl residue revealed that in both positions the tryptophan resided in polar, conformationally heterogeneous environments. The environment of Trp-57 was affected by Ca2+ binding to a much greater extent compared to that of Trp-65. Only 1 equiv of Ca2+ was required to produce greater than 70% of the Trp fluorescence changes in positions 57 and 65, indicating that Ca2+ binding to the higher affinity EF-loop had a pronounced effect on the protein structure.(ABSTRACT TRUNCATED AT 250 WORDS)

'Multifrequency' location and clustering of sequence patterns from proteins.

Comput Appl Biosci. 1991; 7: 31-8

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In previous work, we have shown that a set of characteristics, defined as (code frequency) pairs, can be derived from a protein family by the use of a signal-processing method. This method enables the location and extraction of sequence patterns by taking into account each (code frequency) pair individually. In the present paper, we propose to extend this method in order to detect and visualize patterns by taking into account several pairs simultaneously. Two 'multifrequency' methods are described. The first one is based on a rewriting of the sequences with new symbols which summarize the frequency information. The second method is based on a clustering of the patterns associated with each pair. Both methods lead to the definition of significant consensus sequences. Some results obtained with calcium-binding proteins and serine proteases are also discussed.

Carbohydrate-binding specificity of calcyclin and its expression in human tissues and leukemic cells.

Arch Biochem Biophys. 1991; 289: 137-44

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Binding of biotinylated fetuin in a solid-phase assay served as activity assay for purification of calcyclin, the product of a cell growth-related cDNA with homologies to Ca(2+)-binding proteins. Asialofetuin failed to bind to calcyclin, emphasizing the importance of sialic acids. Binding of fetuin was most effectively reduced by N-glycolylneuraminic acid within a panel of mostly negatively charged sugars. Bovine submaxillary mucin and the ganglioside GM1, but not asialo-GM1, proved more effective than neoglycoproteins, carrying negatively charged carbohydrate moieties. Extension of N-acetyl-neuraminic acid to its lactosyl derivative increased its inhibitory potency. Among charge-free carbohydrate residues, only N-acetylglucosamine, lactose, and mannose, but not fucose, melibiose, or N-acetylgalactosamine affected fetuin binding, substantiating the inherent selectivity. Chemical modification with group-specific reagents revealed that lysine and arginine residues appear to be involved in ligand binding that is optimal in the presence of Ca2+, but not Zn2+ and stable up to 1 m NaCl. Biotinylation of calcyclin by modification of carboxyl groups facilitated performance of solid-phase assays with calcyclin in solution, yielding similar results with (neo)glycoproteins in relation to assays with immobilized calcyclin, thereby excluding an impact of binding to nitrocellulose on calcyclin's specificity. Subcellular fractionation disclosed the presence of fetuin-binding activity in all fractions, the specific activity decreasing from the nuclear to the particulate cytoplasmic fraction and the cytoplasmic supernatant. Affinity-purified antibodies were employed to detect high levels of calcyclin expression in acute lymphoblastic, myelogenous, and monocytic leukemia cell lines, but not in myeloma or lymphoblastoid cells. In comparison, most cells were nearly devoid of an O-acetylsialic acid-specific protein that is more abundant in various tissue types than calcyclin.

The p11 protein is a member of the S-100 family of Ca(2+)-binding proteins and serves within the cell as a ligand of the tyrosine kinase substrate, annexin II. To obtain more structural information on this molecule, we have isolated and characterized p11 cDNA clones from several different species. A comparison of the deduced amino acid (aa) sequences reveals that mammalian and avian p11 are highly similar (at least 90% identical at the aa level), whereas p11 from Xenopus laevis shows a considerable degree of sequence variation (the aa sequence identity drops to approx. 60% when compared to mammalian or chicken p11). Interestingly, the C-terminal 18 aa, which are unique to p11 within the S-100 family, show a relatively high conservation among species. This high evolutionary conservation is in line with a structurally and/or functionally important role of this C terminus, e.g., in annexin II binding.

A set of accurate experimental data is provided for Ca2+ ion binding to calbindin D9k, a protein in the calmodulin superfamily of intracellular regulatory proteins. The study comprises both the role of protein surface charges and the effects of added electrolyte. The two macroscopic Ca2(+)-binding constants K1 and K2 are determined for the wild-type and eight mutant calbindins in 0, 0.05, 0.10, and 0.15 M KCl from titrations in the presence of Quin 2 or 5,5'-Br2BAPTA. The mutations involve replacement of surface carboxylates (of Glu17, Asp19, Glu26, and Glu60) with the corresponding amides. It is found that K1K2 may decrease by a factor of up to 2.5 x 10(5) (triple mutant in 0.15 M KCl as compared to the wild-type protein in 0 M KCl). Ca2(+)-binding constants of the individual Ca2+ sites (microscopic binding constants) have also been determined. The positive cooperativity of Ca2+ binding, previously observed at low salt concentration [Linse et al. (1987) Biochemistry 26, 6723-6735], is also present at physiological ionic strength and amounts to 5 kJ.mol-1 at 0.15 M KCl. The electrolyte concentration and some of the mutations are found to affect the cooperativity. 39K NMR studies show that K+ binds weakly to calbindin. Two-dimensional 1H NMR studies show, however, that potassium binding does not change the protein conformation, and the large effect of KCl on the Ca2+ affinity is thus of unspecific nature. Two-dimensional 1H NMR has also been used to assess the structural consequences of the mutations through assignments of the backbone NH and C alpha H resonances of six mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

The local motion of Tyr13 in wild type and mutant calbindin (Mr 8500, 75 amino acids) was investigated by time-resolved fluorescence spectroscopy performed at the MAX synchrotron in Lund, Sweden. Two-dimensional fluorescence spectroscopy (excitation-emission mapping) was used to characterize the emission of Tyr13 against the background of phenylalanine residues in the presence and absence of Ca2+. Local restricted motion of Tyr13 is observed in wild-type calbindin with only minor differences between the Ca2(+)-saturated and Ca2(+)-free forms. In a mutant, where Pro20 is exchanged for Gly and Ala14 and Asn21 are deleted, the local mobility of Tyr13 is enhanced close to values characteristic for free rotational diffusion. An increase of the overall rotational motion in this mutant form by a factor of two and the enhanced local mobility of Tyr13 indicate local and global conformational changes that also affect the Ca2(+)-binding properties. Tyr13 occurs in two isomeric species differing in lifetime of the excited state; the major species is populated to 85-90%.

Studies on the structural properties of lipocortin-1 and the regulation of its synthesis by steroids.

Prog Clin Biol Res. 1990; 349: 27-45

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Lipocortin-1 protein synthesis in resting monocytes is under the control of glucocorticoid steroids. This induction occurs at reasonable dexamethasone concentrations, may require concomitant synthesis of transcriptional factors, appears to be cell type specific, and has been observed only in primary tissues in our hands. Variability in the magnitude of the induction suggests that the regulation is complex, involving either additional factors or particular differentiation states. In addition to the induction of intracellular lipocortin-1, steroids cause the appearance of labelled lipocortin-1 on the outer surface of the cells. Whether cell breakage can account for this effect is unclear. Considerable microheterogeneity was found in preparations of recombinant-lipocortin-1. Aspects of N-terminal post-translational processing, N-terminal proteolysis, conformational states and the existence of an air-denatured form lacking alpha-helical structure contributed to this heterogeneity. We believe that these aspects are responsible for the variable biological potency of different preparations. It remains unclear whether this protein actually plays a physiological role in the regulation of the inflammatory response or achieves its effects through membrane binding and subsequent non-physiological perturbation of the cells.

Ca2(+)-binding site of carp parvalbumin recognized by monoclonal antibody.

Cell Calcium. 1990; 11: 19-23

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Monoclonal antibody 235 which was used for immunohistochemical staining of parvalbumin in tissue sections partially protects Lys-54 of carp muscle parvalbumin from reaction with acetic anhydride in the parvalbumin-antibody complex. Lys-54 is located in the CD-loop of parvalbumin and is flanked by the Ca2(+)-ligands Asp-53 and Ser-55 of the Ca2(+)-site I. Another monoclonal antibody against carp parvalbumin, mca 239, partially protects lysine residues 27, 32, 87 and 107, indicating that this antibody is directed against a discontinuous epitope distant from the two Ca2(+)-binding sites of parvalbumin.

Comparison of metal ion-induced conformational changes in parvalbumin and oncomodulin as probed by the intrinsic fluorescence of tryptophan 102.

J Biol Chem. 1990; 265: 11456-64

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The calcium-induced conformational changes of the 108-amino acid residue proteins, cod III parvalbumin and oncomodulin, were compared using tryptophan as a sensitive spectroscopic probe. As native oncomodulin is devoid of tryptophan, site-specific mutagenesis was performed to create a mutant protein in which tryptophan was placed in the identical position (residue 102) as the single tryptophan residue in cod III parvalbumin. The results showed that in the region probed by tryptophan-102, cod III parvalbumin experienced significantly greater changes in conformation upon decalcification compared to the oncomodulin mutant, F102W. Addition of 1 eq of Ca2+ produced greater than 90% of the total fluorescence response in F102W, while in cod III parvalbumin, only 74% of the total was observed. Cod III parvalbumin displayed a negligible response upon Mg2+ addition. In contrast, F102W did respond to Mg2+, but the response was considerably less when compared to Ca2+ addition. Time-resolved fluorescence showed that the tryptophan in both proteins existed in at least two conformational states in the presence of Ca2+ and at least three conformational states in its absence. Comparison with quantum yield measurements indicated that the local electronic environment of the tryptophan was significantly different in the two proteins. Collectively, these results demonstrate that both cod III parvalbumin and oncomodulin undergo Ca2(+)-specific conformational changes. However, oncomodulin is distinct from cod III parvalbumin in terms of the electronic environment of the hydrophobic core, the magnitude of the Ca2(+)-induced conformational changes, and the number of calcium ions required to modulate the major conformational changes.

The protochordate amphioxus (Branchiostoma lanceolatum) contains different isoforms of sarcoplasmic, high-affinity Ca2(+)-binding proteins (SCP). The amino acid sequences of the two major isoforms SCP I and II, reported previously [Takagi, T., Konishi, K. & Cox, J.A. (1986) Biochemistry 25, 3585-3592], have been corrected and differ from each other by seven amino acid substitutions in a 17-residue-long segment (positions 20-36). We also report on the isolation and amino acid sequence determination of two minor isoforms, i.e. amphioxus SCP III and IV. Although they behave very differently from the major forms with respect to net charge, they differ from SCP I by only one amino acid: SCP III has Met at position 20 (Tyr in SCP I) and SCP IV has Asn at position 23 (Asp in SCP I). Together the sequence data on amphioxus SCP suggest that, in contrast with SCP of other invertebrate phyla, the isoforms are generated by alternative splicing of the primary RNA transcript with a mutually exclusive pattern.

We have constructed three different engineered proteins based on calbindin D9K by either exchanging the two calcium binding sites within the protein or making the amino acid sequence of the two calcium binding sites identical. The individual calcium binding sites of the engineered proteins retain most of their ion binding characteristics as well as the basal structure of their Ca2+ ligand sphere in the new environment. Even the protein with its sites interchanged, a mutation involving 30 amino acids out of a total of 75, still binds calcium with an affinity as high as that of many natural EF-hand proteins.

Comparative structural analysis of the calcium free and bound states of the calcium regulatory protein calbindin D9K.

J Mol Biol. 1990; 213: 593-8

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The solution structure of apo calbindin D9K, a member of the calmodulin superfamily of calcium-binding regulatory proteins, has been investigated by 1H nuclear magnetic resonance spectroscopy and the results compared with a corresponding study of the calcium-loaded protein. On the basis of complete sequence-specific assignments, characteristic patterns of short proton-proton distances have been identified in two-dimensional nuclear Overhauser effect spectra, allowing the elements of secondary structure to be determined. It is found that four helices and a short section of antiparallel beta-sheet are present regardless of the calcium content of the protein. In addition, a preliminary analysis of the long-range nuclear Overhauser effects shows that the global folding patterns are the same and that the tertiary structures of the apo protein is very similar to that of the calcium-loaded protein. These results are in stark contrast to a number of very substantial changes in 1H chemical shift. Preliminary studies of protein dynamics show some very large differences in flexibility and internal mobility. This suggests that protein dynamics may play a role more important than was initially realized in the function of calbindin D9K and other homologous calcium-binding regulatory proteins.

Ca(2+)-ions play a key role in the regulation of many cellular processes and impairment of calcium homeostasis has been implicated in several diseases. Intracellularly the Ca(2+)-signal is transmitted by two families of proteins, the 'EF-hand'- and the Ca(2+)-dependent and phospholipid binding proteins. Their protein and gene structures as well as possible functional roles are summarized.

The complete amino acid sequence of the Ca2(+)-triggered luciferin binding protein (LBP) of Renilla reniformis has been determined. The apoprotein has an unblocked amino terminus and contains 184 residues with a calculated Mr of 20,541. LBP is a member of the EF-hand superfamily of Ca2(+)-binding proteins and bears three predicted EF-hand domains. The sequence and organization of EF-hand domains are similar to those of the Ca2(+)-dependent photoprotein, aequorin.

Expression of immunoreactivity for Ca-binding protein, spot 35 in the interstitial cell of the rat pineal organ.

Histochem J. 1990; 22: 4-10

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In the rat pineal organ numerous stellate cells exhibited intense immunoreactivity for calcium-binding spot 35 protein. Because of their peculiar shape and ultrastructure, identical to those of intrapineal S-100-immunoreactive cells, the spot 35-immunoreactive stellate cells were identified as the interstitial cells. The comparison of the morphology and population density of spot 35-, S-100-, and GFAP (glial fibrillar acidic protein)-immunoreactive cells, suggests that spot 35-immunoreactive cells represent a major subpopulation of the interstitial cells, all of which are S-100-immunoreactive and generally considered to be of glial nature, while GFAP-immunoreactive cells represent a minor subpopulation of the interstitial cells located in the proximal part close to the pineal stalk. This is the first report describing the occurrence of the calcium-binding protein in cells of glial nature.

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.

Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments.

Biochem Cell Biol. 1990; 68: 587-601

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This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.

p36 (annexin 2) is the major cytoplasmic target of the src tyrosine-kinase and forms in vitro and in vivo a stable tetrameric complex in which two p36 polypeptides interact with a dimer of a unique p11 polypeptide. p11 belongs into the superfamily of EF-hand proteins. Upon mild cysteine modification conditions, both cysteines (position 61 and 82) of the free p11 become substituted, and the ability to form the p36.p11 complex is lost. Under the same conditions, the 2 cysteines of p11 incorporated into the complex display differential reactivity. Here, cysteine 61 is fully substituted while cysteine-82 is protected. p11 derivatives substituted only on cysteine 61 retain binding activity for p36 unless cysteine 82 is substituted by a second cycle of modification of the isolated p11. Thus, the C-terminal extension protruding from the second EF-hand of the p11 molecule (residues 77-96) is important for the interaction with p36. As a consequence of our analysis, we report a new separation of p36 and p11 from the p36.p11 complex. This is based on a reversible cysteine modification and thus is an alternative to the denaturation and renaturation cycle used previously.

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.

One- and two-dimensional 1H NMR have been used to study the backbone dynamics in Ca2(+)-free (apo) and Ca2(+)-loaded (Ca2) calbindin D9k at pH 7.5 and 25 degrees C. Hydrogen exchange rates of all 71 backbone amide protons (NH's) have been measured for the Ca2 form by both a direct exchange-out experiment and another experiment that measures the transfer of saturation from water protons to amide protons. A large number of NH's are found to be highly protected against exchange with solvent protons. The results for the Ca2 form are related to solvent accessibility and hydrogen bonding obtained in molecular dynamics simulations of calcium-loaded calbindin. The correlation with these parameters is strong within the N-terminal half of calbindin, which is found to be more stable than the C-terminal half. The amide proton exchange in the apo form is much faster than in the Ca2 form and was studied in a series of experiments in which the exchange was quenched after different times by Ca2+ addition. This experiment is applicable to all amide hydrogens that exchange slowly in the Ca2 form. For these NH's the effects of Ca2+ removal span from a 10(2)-fold decrease to a 10(5)-fold increase of the exchange rate, and the average is a 220-fold increase. The effects on individual NH exchange rates show that the four alpha-helices are almost intact after calcium removal and that the changes in dynamics involve not only the Ca2(+)-binding region. Hydrogen bonds involving backbone NH's in the Ca2+ loops appear to be broken or weakened when calbindin releases Ca2+, whereas the beta-sheet between the Ca2+ loops is found to be present in both the Ca2 and apo forms. Large Ca2(+)-induced effects on NH exchange rates were measured for a few residues at alpha-helix ends far from the two Ca2(+)-binding sites. This may be the result of a change in interhelix angles (or the rate of interhelix angle fluctuations) on calcium binding.

Protein-protein recognition via short amphiphilic helices; a mutational analysis of the binding site of annexin II for p11.

EMBO J. 1990; 9: 4207-13

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Annexin II (p36) interacts with its ligand p11 via the short stretch of 12 amino acids (Ac-S-T-V-H-E-I-L-C-K-L-S-L) situated at the N-terminus. We have now synthesized some 37 tetradecapeptides, which differ from the original p11 binding sequence (Ac1-14) by single amino acid substitutions. The relative affinity of each peptide for p11 was determined by fluorescence spectroscopy using a competitive binding assay. The binding behaviour of the different peptides confirms the model of an amphiphilic alpha-helix induced upon binding to p11. The apparent affinities delta delta Gbind of the mutant peptides revealed that the N-acetyl group of serine 1 and the hydrophobic side chains at positions 3, 6, 7 and 10 contribute most to the binding. The observed destabilization of the complex upon removal of signal methyl groups from the hydrophobic side of the helix is comparable with the destabilization of proteins in which methyl groups have been removed from the inner core. We conclude that upon binding to p11 the hydrophobic side of the amphiphatic alpha-helix becomes fully buried.

Interaction of two brain annexins, CaBP33 and CaBP37, with membrane-skeleton proteins.

FEBS Lett. 1990; 267: 171-5

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CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X-100-resistant fraction (cytoskeleton) from bovine brain membranes in a Ca2(+)-dependent way in vitro. The binding is saturable with respect to the CaBP33-CaBP37 concentration, half-maximal binding occurring at approximately 15 micrograms of the CaBP33-CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca2+ concentration is biphasic, with half-maximal binding at approximately 50 microM and approximately 400 microM free Ca2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low Mr polypeptides (10-20 kDa) in the crude cytoskeleton preparation, with formation of an 85-90 kDa complex as investigated in cross-linking experiments. No binding of the CaBP33-CaBP37 mixture to either G- or F-actin has been observed. Identification of the CaBP33-CaBP37-binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.

19F-n.m.r. spectra were measured to investigate the effects of Ca2+ and Zn2+ on the interaction of trifluoperazine (TFP) with three S100 proteins. It was found that TFP binds to S100a and S100ao proteins irrespective of the presence of Ca2+ and Zn2+, while in the presence of Ca2+ the apparent affinity of TFP to the proteins was greater than that in its absence or in the presence of Zn2+. In contrast, the binding affinity of TRP to S100b protein in the presence and absence of metal ions was lower than to S100a and S100ao proteins. These results suggested that TFP binds to each S100 protein in two ways: one is Ca2(+)- or Zn2(+)-dependent specific manner and another is Ca2(+)- or Zn2(+)-independent non-specific manner.

The third calmodulin family protein in Tetrahymena. Cloning of the cDNA for Tetrahymena calcium-binding protein of 23 kDa (TCBP-23).

J Biol Chem. 1990; 265: 2514-7

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Recently, we proved the existence of the second calmodulin family protein in Tetrahymena (Tetrahymena calcium-binding protein of 25 kDa, TCBP-25) by analyzing its cDNA (Takemasa, T., Ohnishi, K., Kobayashi, T., Takagi, T., Konishi, K., and Watanabe, Y. (1989) J. Biol. Chem. 264, 19293-19301). During the amino acid sequence determination of TCBP-25, we became aware of the fact that another polypeptide carrying calcium-binding domains of EF-hand type existed in addition to Tetrahymena calmodulin and TCBP-25. This third calmodulin family protein from Tetrahymena was confirmed by isolating its cDNA clones. One of the cloned cDNAs contains 763 nucleotides and encodes a protein that is composed of 207 amino acid residues and has a molecular mass of 23,413 daltons. This predicted protein possesses four EF-hand type calcium-binding domains, so we have designated it as Tetrahymena calcium-binding protein of 23 kDa (TCBP-23). TCBP-23 is similar (35% homology) but clearly different from TCBP-25. The TCBP-23 gene is actively transcribed in vivo as a 0.84-kilobase RNA. Thus, it follows that Tetrahymena cells have three different calmodulin family proteins: calmodulin, TCBP-25 and TCBP-23. These proteins are expected to provide important clues for solving the mechanisms of calcium-dependent phenomena, such as ciliary reversal.

The solution structure and dynamics of apo bovine calbindin D9k have been studied by a wide range of two-dimensional 1H nuclear magnetic resonance experiments. Due to the presence of conformational heterogeneity in the wild-type protein, the sequential resonance assignment was carried out on a Pro43----Gly mutant. By use of a combination of scalar correlation experiments acquired from H2O solution, 61 of the 76 1H spin systems could be assigned to particular amino acid types. The remaining resonances were assigned by a parallel series of experiments acquired from 2H2O solution. These spin system assignments provided a basis for complete sequential resonance assignments from interresidue backbone nuclear Overhauser effects (NOEs). Elements of secondary structure were identified from sequential and medium-range NOEs, backbone spin-spin coupling constants, and slowly exchanging amide protons. Four sections of helix are delineated, together with a short antiparallel beta-sheet interaction between the peptide loops involved in Ca2+ binding. The global fold is provided by combining these elements of secondary structure with a subset of the long-range, interhelix NOEs. Comparison with similar studies on the Ca2(+)-saturated protein indicates that at this crude level the structures are very similar. However, removal of the Ca2+ does dramatically affect the dynamics of the protein, as judged by amide proton exchange rates and aromatic ring rotation. This is particularly evident in the increased flexibility of the residues in the hydrophobic core.

Interaction of metal ions with carboxylic and carboxamide groups in protein structures.

Protein Eng. 1990; 4: 49-56

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An analysis of the geometry of metal binding by carboxylic and carboxamide groups in proteins is presented. Most of the ligands are from aspartic and glutamic acid side chains. Water molecules bound to carboxylate anions are known to interact with oxygen lone-pairs. However, metal ions are also found to approach the carboxylate group along the C-O direction. More metal ions are found to be along the syn than the anti lone-pair direction. This seems to be the result of the stability of the five-membered ring that is formed by the carboxylate anion hydrogen bonded to a ligand water molecule and the metal ion in the syn position. Ligand residues are usually from the helix, turn or regions with no regular secondary structure. Because of the steric interactions associated with bringing all the ligands around a metal center, a calcium ion can bind only near the ends of a helix; a metal, like zinc, with a low coordination number, can bind anywhere in the helix. Based on the analysis of the positions of water molecules in the metal coordination sphere, the sequence of the EF hand (a calcium-binding structure) is discussed.

Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard. Both structural and functional studies establish that 67-kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67-kDa calcimedin with the predicted sequence of 67-kDa calelectrin, we concluded that the primary structure of these 67-kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family. A 34-kDa fragment of 67-kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross-reactivity. The sequence data further support that the 34-kDa fragment is derived from the C-terminal portion of 67-kDa calcimedin by limited proteolysis. The 34-kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F-actin and phospholipids.

It has been found in in vitro experiments that fluorescence intensity of deionized solution containing a chlorotetracycline fluorescent probe increases insignificantly at the addition of calmodulin of S-100 proteins. Subsequent introduction of Ca2+ into the medium results in the pronounced fluorescence increase depending on Ca2+ concentration. Addition of specific protein blockers--W7 (calmodulin inhibitor) and antibodies to S-100 brought about a decrease of fluorescence. In in vivo experiments on chlorotetracycline-stained neurons of Helix Pomatia ganglia subesophageal complex it has been shown that bringing of antibodies to S-100 and calmodulin significantly decreases the fluorescence intensity of these cells. These data suggest that the chlorotetracycline probe is an indicator of calcium ions binding with calcium-binding proteins both in in vitro and in vivo systems.

Purification and characterization of annexin proteins from bovine lung.

Biochemistry. 1990; 29: 4852-62

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Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.

Purification and characterization of an abundant cytosolic protein from human neutrophils that promotes Ca2(+)-dependent aggregation of isolated specific granules.

J Clin Invest. 1990; 85: 1065-71

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Intracellular ionized calcium has been strongly implicated in mediating several responses of human neutrophils to stimulation. However, proteins that serve as effectors of these responses have not been well characterized. To identify proteins that might serve as mediators of the effects of Ca2+ in human neutrophils, we isolated proteins that bind to membrane phospholipids in a Ca2(+)-dependent manner. The most abundant of these, a protein of 33 kD, was readily purified to homogeneity, and was found to bind to phosphatidylserine vesicles in the presence of 2 microM ionized Ca2+. In addition, this purified protein promoted Ca2(+)-dependent aggregation of isolated specific granules from human neutrophils, indicating that it might mediate membrane-membrane contact during processes such as phagosome-lysosome fusion or degranulation. This protein was localized to the cytoplasm of unstimulated neutrophils and found to account for approximately 1% of the cytosol protein. Amino acid sequence of several peptides derived from the purified protein revealed that it is identical to lipocortin III, a recently described member of the annexin family that is scarce in other cells and tissues. The abundance of this protein, together with its Ca2(+)-dependent membrane effects, suggest that it mediates membrane-localized events in stimulated neutrophils, such as phagosome-lysosome fusion or degranulation.

The calcium-binding protein calbindin D9k has previously been shown to exist in two folded forms only differing in the proline cis-trans isomerism of the Gly-42-Pro-43 amide bond. This bond is located in a flexible loop connecting the two EF-hand Ca2+ sites. Calbindin D9k therefore constitutes a unique test case for investigating if the recently discovered enzyme peptidyl-prolyl cis-trans isomerase (PPIase) can affect the cis-trans exchange rate in a folded protein. The 1H NMR saturation transfer technique has been used to measure the rate of interconversion between the cis and trans forms of calbindin in the presence of PPIase (PPIase:calbindin concentration ratio 1:10) at 35 degrees C. No rate enhancement could be detected.

Stopped-flow fluorescence kinetic measurements, circular dichroism (CD), and 1H nuclear magnetic resonance (NMR) spectroscopy at 360 MHz have been used to study the interaction of the calcium-channel blocker and calmodulin antagonist bepridil with cardiac troponin C (cTnC) in the presence of calcium. The kinetic data show that bepridil reduces the rate of calcium release only from the low affinity, calcium-specific site and not from the two high affinity calcium/magnesium sites. CD measurements indicate that drug binding leads to a small increase in the alpha-helical content of the complex. 1H NMR shows that the protein binds one equivalent of bepridil, with a dissociation constant of approximately 20 microM, only when the low affinity calcium site is occupied. Exchange is fast or intermediate on the chemical shift time scale. Drug binding is shown to be largely localized in the N-terminal domain, containing the low affinity calcium site, by observing the shifting and broadening of several resonances associated with that domain. These include assigned aromatic signals together with methionyl and other methyl signals. Observation of intermolecular nuclear Overhauser effects was precluded by extensive spectral overlap. Consideration of the data from the three techniques permitted a model of the bepridil-cTnC complex to be constructed, using the model of cTnC derived from the x-ray structure of calmodulin (MacLachlan L. K., Reid, D. G., and Carter, N. (1990) J. Biol. Chem. 265, 9754-9763). Binding of bepridil to a prominent hydrophobic depression in the N-terminal domain can be invoked to explain many of the induced changes in the spectral and kinetic properties of the protein. The implications of the model for the calcium sensitizing action of bepridil are discussed.

Proton (1H) NMR at 360 MHz has been used to characterize calcium-induced spectral changes in bovine cardiac troponin C in more detail than hitherto reported (Hincke, M. T., Sykes, B. D., and Kay, C. M. (1981) Biochemistry 20, 3286-3294). The observed changes are consistent with two equivalents of calcium occupying high affinity sites, with subsequent binding of a single equivalent to a lower affinity site. Two-dimensional J-correlated and nuclear Overhauser effect NOE-correlated and conventional one-dimensional NOE experiments, combined with a consideration of the titration behavior, have allowed all the aromatic signals, and several prominently shifted alpha-CH and methyl group signals, as well as some methionine methyl signals of the calcium-saturated protein, to be assigned. This exercise was facilitated by the construction of a model of the calcium-bound protein based on crystal structure data of the homologous calmodulin and skeletal troponin C, using mutations, energy minimizations, and molecular dynamics simulations, combined with the ring-current shift and NOE prediction program PARSNIP (Reid, D. G., and Saunders, M. R. (1989) J. Biol. Chem. 264, 2003-2012).

Eu3+ luminescence studies of oncomodulin. The origin of the pH-dependent behavior.

J Biol Chem. 1990; 265: 9694-700

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The Eu3+ 7F0----5D0 excitation spectra of parvalbumin and oncomodulin are pH-dependent. Until now, it had been assumed that both the CD and EF ion-binding sites shared this property and that deprotonation of water molecules coordinated to the bound Eu3+ ions might be responsible for the pH dependence. Results obtained with the site-specific variant of oncomodulin known as D59E, in which glutamate replaces the aspartate naturally present at position 59, have necessitated substantial revision of these ideas. It now appears that the pH-dependent behavior is confined to the CD site. Moreover, we observe no corresponding change in the number of O-H oscillators coordinated to the bound Eu3+ ions in the pH range over which we observe the spectroscopic alteration. It is likely that the behavior results from deprotonation of one or more carboxyl groups clustered at the COOH-terminal end of the CD domain.

Calcium(II) site specificity: effect of size and charge on metal ion binding to an EF-hand-like site.

Biochemistry. 1990; 29: 3937-43

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The molecular mechanisms by which protein Ca(II) sites selectively bind Ca(II) even in the presence of high concentrations of other metals, particularly Na(I), K(I), and Mg(II), have not been fully described. The single Ca(II) site of the Escherichia coli receptor for D-galactose and D-glucose (GGR) is structurally related to the eukaryotic EF-hand Ca(II) sites and is ideally suited as a model for understanding the structural and electrostatic basis of Ca(II) specificity. Metal binding to the bacterial site was monitored by a Tb(III) phosphorescence assay: Ca(II) in the site was replaced with Tb(III), which was then selectively excited by energy transfer from protein tryptophans. Photons emitted from the bound Tb(III) enabled specific detection of this substrate; for other metals binding was detected by competitive displacement of Tb(III). Representative spherical metal ions from groups IA, IIA, and IIIA and the lanthanides were chosen to study the effects of metal ion size and charge on the affinity of metal binding. A dissociation constant was measured for each metal, yielding a range of KD's spanning over 6 orders of magnitude. Monovalent metal ions of group IA exhibited very low affinities. Divalent group IIA metal ions exhibited affinities related to their size, with optimal binding at an effective ionic radius between those of Mg(II) (0.81 A) and Ca(II) (1.06 A). Trivalent metal ions of group IIIA and the lanthanides also exhibited size-dependent affinities, with an optimal effective ionic radius between those of Sc(III) (0.81 A) and Yb(III) (0.925 A). The results indicate that the GGR site selects metal ions on the basis of both charge and size.(ABSTRACT TRUNCATED AT 250 WORDS)

Pancreatic thread protein (PTP) forms double helical threads in the neutral pH range after purification, undergoing freely reversible, pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Gross et al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing greater than 99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH2-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca2(+)-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.

The relationships among 153 EF-hand (calcium-modulated) proteins of known amino acid sequence were determined using the method of maximum parsimony. These proteins can be ordered into 12 distinct subfamilies--calmodulin, troponin C, essential light chain of myosin, regulatory light chain, sarcoplasmic calcium binding protein, calpain, aequorin, Stronglyocentrotus purpuratus ectodermal protein, calbindin 28 kd, parvalbumin, alpha-actinin, and S100/intestinal calcium-binding protein. Eight individual proteins--calcineurin B from Bos, troponin C from Astacus, calcium vector protein from Branchiostoma, caltractin from Chlamydomonas, cell-division-cycle 31 gene product from Saccharomyces, 10-kd calcium-binding protein from Tetrahymena, LPS1 eight-domain protein from Lytechinus, and calcium-binding protein from Streptomyces--are tentatively identified as unique; that is, each may be the sole representative of another subfamily. We present dendrograms showing the relationships among the subfamilies and uniques as well as dendrograms showing relationships within each subfamily. The EF-hand proteins have been characterized from a broad range of organismal sources, and they have an enormous range of function. This is reflected in the complexity of the dendrograms. At this time we urge caution in assigning a simple scheme of gene duplications to account for the evolution of the 600 EF-hand domains of known sequence.

Biophysical studies of engineered mutant proteins based on calbindin D9k modified in the pseudo EF-hand.

Eur J Biochem. 1990; 187: 455-60

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The genes for four mutant proteins from calbindin D9k, all with mutations in the N-terminal Ca2+-binding domain (pseudo EF-hand) have been synthesized and expressed in Escherichia coli. The purification scheme has been modified to minimize the formation of deamidated proteins. The set of modifications in the pseudo EF-hand is an attempt to turn this site into a structure resembling an archetypal EF-hand, with its characteristic 113Cd-NMR shift (-80 to -110 ppm) and high calcium-binding constants, whereas the C-terminal Ca2(+)-binding site (EF-hand) is kept intact in all mutant proteins. The mutant proteins studied here all have pseudo EF-hands with a lower calcium-binding constant and a higher calcium off-rate to the pseudo EF-hand than the wild-type protein. From the results obtained it is obvious that proline 20 in the pseudo EF-hand, which has been deleted or replaced by glycine in three of the mutants, has a stabilizing effect on calcium binding to that site. Furthermore, the modifications in the pseudo EF-hand seem to have only a local effect, leaving the tertiary structure of the protein and the calcium-binding properties of the unmodified site virtually unchanged.

The Rhizobium nodulation gene nodO encodes a Ca2(+)-binding protein that is exported without N-terminal cleavage and is homologous to haemolysin and related proteins.

EMBO J. 1990; 9: 349-54

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Nodulation and host-specific recognition of legumes such as peas and Vicia spp. are encoded by the nodulation (nod) genes of Rhizobium leguminosarum biovar viciae. One of these genes, nodO, has been shown to encode an exported protein that contains a multiple tandem repeat of a nine amino acid domain. This domain was found to be homologous to repeated sequences in a group of bacterial exported proteins that includes haemolysin, cyclolysin, leukotoxin and two proteases. These proteins are secreted by a mechanism that does not involve an N-terminal signal peptide. The NodO protein is present in the growth medium of Rhizobium bacteria induced for nod gene expression, and partial protein sequencing of the purified protein showed that there is no N-terminal cleavage of the exported protein. It has been suggested that the internally repeated domain of haemolysin may be involved in Ca2(+)-mediated binding to erythrocytes and we show that the NodO protein can bind 45Ca2+. It is proposed that the NodO protein may interact directly with plant root cells in a Ca2(+)-dependent way, thereby mediating an early stage in the recognition that occurs between Rhizobium and its host legume.

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes.

J Biol Chem. 1990; 265: 17946-52

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A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.

Organization of polar groups of 9 kd calbindin around Ca2+ ions bound to the protein: a microdielectric study.

Protein Eng. 1990; 4: 121-4

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Using a simple model of linear response of polarizable centers to an electric field, function delta is defined to characterize structural organization of protein polar groups. The function makes it possible to detect specific structures of nonuniformly distributed and mutually coupled groups that can transmit local structural and charge density perturbations, induced by an ion over long distances to functionally active sites of a protein molecule. In 9 kd calbinding (a small protein from the troponin C superfamily) two structural chains have been demonstrated that link together both Ca2+ ions coordinated by the protein. The chains form a rigid structure stabilized by coordination of one of the ions, so that binding of the other is promoted. Such a structure is probably common to all members of the superfamily and plays an important role in the mechanism of calcium binding by these proteins.

Two-dimensional nuclear magnetic resonance spectra of porcine C5adesArg (73 residues) have been used to construct a list of 34 hydrogen bonds, 27 dihedral angle constraints, and 151 distance constraints, derived from nuclear Overhauser effect data. These constraints were used in restrained molecular dynamics calculations on residues 1-65 of C5a, starting from a folded structure modeled on the crystal structure of a homologous protein, C3a. Forty-one structures have been calculated, which fall into three similar families with few violations of the imposed constraints. Structures in the most populated family have a root-mean-square deviation from the average structure of 1.02 A for the C alpha atoms, with good definition of the internal residues. There is good agreement between the calculated structures and other nuclear magnetic resonance data. The structure is very similar to that recently reported for human C5a [Zuiderweg et al. (1989) Biochemistry 28, 172-185]. Some biological implications of these structures are discussed.

Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.

Biochem J. 1990; 271: 473-80

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In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.

Terbium luminescence in synthetic peptide loops from calcium-binding proteins with different energy donors.

J Biol Chem. 1990; 265: 10358-66

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Fourteen 14-mer peptides corresponding to a consensus sequence of metal-binding loops from proteins of the calmodulin family were synthesized. The effect of varying both the position in the binding loop, and the type of aromatic side chains as energy donors for enhancement of terbium luminescence, was studied. It was concluded that tryptophan in loop position 7 gave optimal luminescence enhancement, and that the additional inclusion of a tyrosine in the loop at positions 2 or 4 could further boost emission from the bound terbium. In all further cases energy transfer from aromatic residues at positions other than 7 was markedly less efficient. These results suggest that the peptides assume a configuration which allows a hexadentate ligand structure around the bound terbium ion. This is consistent with a Dexter-type electron exchange model of energy transfer.

The kinetics of calcium dissociation from two groups of site-specific mutants of calbindin D9k--a protein in the calmodulin superfamily with two Ca2+ sites and a tertiary structure closely similar to that of the globular domains of troponin C and calmodulin--have been studied by stopped-flow kinetic methods, using the fluorescent calcium chelator Quin 2, and by 43Ca NMR methods. The first group of mutants comprises all possible single, double, and triple neutralizations of three particular carboxylate groups (Glu-17, Asp-19, and Glu-26) that are located on the surface of the protein. These carboxylates are close to the two EF-hand calcium binding sites, but are not directly liganded to the Ca2+ ions. Conservative modification of these negative carboxylate side chains by conversion to the corresponding amides results in a marked reduction in the Ca2+ binding constants for both sites, as recently reported [Linse et al. (1988) Nature 335, 651-652]. The stopped-flow kinetic results show that this reduction in Ca2+ affinity derives primarily from a reduction in the Ca2+ association rate constant, kon. The estimated maximum value of the association rate constant (kon(max) for Ca2+ binding to the wild-type protein is ca. 10(9) M-1 s-1. In contrast, for the mutant protein with three charges neutralized the maximum association rate constant is estimated to be only 2 X 10(7) M-1 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant.

Biochemistry. 1990; 29: 4400-9

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The EF-hand calcium-binding protein, calbindin D9k, exists in solution in the calcium-loaded state, as a 1:3 equilibrium mixture of two isoforms, the result of cis-trans isomerism at the Gly42-Pro43 peptide bond [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198]. Nuclear magnetic resonance (NMR) studies of the minor (cis-Pro43) isoform and the Pro43----Gly mutant are reported here. The rate of cis----trans isomerization at the Pro43 peptide bond in the wild-type protein was determined by line-shape analysis at elevated temperatures, using a sample in which all amino acids, except Ser and Val, were deuterated. The cis----trans rate is calculated to be 0.2 s-1 at 25 degrees C, corresponding to a free energy of activation, delta G, of 77 kJ/mol. The complete sequence-specific 1H NMR assignments of the cis-Pro43 isoform and the Pro43----Gly mutant in the calcium-loaded state have been obtained by using standard methods combined with comparisons to the previously assigned major (trans-Pro43) isoform. This has permitted detailed comparative analysis of 1H NMR chemical shifts, backbone scalar coupling constants, and nuclear Overhauser effects. The minor isoform has a global fold that is identical with that of the major isoform. Structural changes imposed by cis-trans isomerization at Pro43 are highly localized to the linker loop (containing Pro43) that joins the two EF hands. The Pro43----Gly mutant has a global fold that is identical with the wild-type protein, but does not exhibit conformational heterogeneity. Only very limited structural differences are observed between mutant and wild-type protein, and these are also highly localized to the linker loop. The ion-binding properties of the mutant, as determined by 43Ca and 113Cd NMR, are found to be very similar to the wild-type protein. These results provide crucial evidence that justifies the calculation of high-resolution three-dimensional structures of the Pro43Gly mutant, rather than of the conformationally heterogeneous wild-type protein.

S100 beta is a multifunctional protein that is found in large amounts in astrocytes and a number of other tissues. In the developing nervous system, S100 beta is secreted from proliferating astrocytes during the time of neurite outgrowth from cortical neurons. The secreted form has neurotrophic activity on primary neurons and neuroblastoma cells. The neurotrophic activity is sensitive to reduction of disulfide bonds, and appears to be a disulfide dimer of S100 beta. The accumulation of S100 beta in mature glial cells is associated with microtubule network. The transfection and expression of cDNA for S100 beta in mammalian cells confers neurotrophic activity on extracts of these cells. Based on our observations of a neurotrophic activity for S100 beta, the occurrence of the gene for S100 beta on human chromosome 21 (Allore et al., 1988) and the elevated levels of S100 beta-containing cells in AD and DS brains (Griffin et al., 1989), we suggest that S100 beta plays a role in the abnormal development of the nervous system in DS and the degeneration of central neurons in AD. It is essential at the present stage to demonstrate the action of S100 beta in vivo, and to construct animal models, such as transgenic mice, that overexpress S100 beta. Such models will allow the dissection of the role of S100 beta in the developing and degenerating central nervous system.

Human placental annexin IV, a member of the annexin family of calcium and phospholipid-binding proteins, has been crystallized by the vapour diffusion method in the presence of calcium, using polyethylene glycol 8000. The crystals are orthorhombic, space C222(1), cell dimensions a = 105.4 A, b = 115.7 A, c = 80.7 A and diffract to at least 2.5 A resolution on a synchrotron source.

Identification of an isoaspartyl linkage formed upon deamidation of bovine calbindin D9k and structural characterization by 2D 1H NMR.

Biochemistry. 1989; 28: 8646-53

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Preparations of recombinant bovine calbindin D9k (r-calbindin) that appear homogeneous on SDS electrophoresis gels have been shown by isoelectric focusing to be mixtures of proteins differing in net charge. The production of two isoforms with increased negative charge occurs during a routine urea denaturation step and can be effectively suppressed by replacing this procedure with thermal denaturation. The two isoforms have been separated from the native protein by DEAE-Sephacel ion-exchange chromatography. Amino acid sequencing of tryptic peptide fragments and two-dimensional (2D) 1H NMR studies establish that the isoforms correspond to calbindin D9k deamidated at Asn56 and that the major product has an isoaspartate (beta-linked peptide) residue at this position. The minor deamidated component is found to have a normal Asp-Gly alpha-linkage. A detailed analysis of proton chemical shifts, phi backbone dihedral angles, and nuclear Overhauser effects indicates that the global conformation of r-calbindin is not perturbed upon deamidation and that all elements of secondary structure are intact. The Asp56 form is nearly identical with the intact protein, whereas the structure of the iso-Asp56 form is perturbed, predominantly in the polypeptide segment Lys55-Asp58. These studies demonstrate that 2D 1H NMR techniques can be used to identify and quantitate the two isoforms produced upon deamidation of a protein and to assess changes in the local and global conformation.

Amino acid sequence of a low molecular weight, high affinity calcium-binding protein from the optic lobe of the squid Loligo pealei.

J Biol Chem. 1989; 264: 7202-9

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The amino acid sequence was determined for squid calcium-binding protein (CaBP), a low molecular weight, high affinity calcium-binding protein from squid optic lobe. The sequence shows this protein to consist of 149 amino acids with an N-acetylated N terminus. The protein has a molecular weight of 16,894 and is homologous to calmodulin, which is also present in squid optic lobe (Head, J. F., Spielberg, S., and Kaminer, B. (1983) Biochem. J. 209, 797-802). When the sequences of squid CaBP and bovine brain calmodulin are appropriately aligned, the proteins are found to share 68% identity, with a single residue insertion in squid CaBP, between domains III and IV. The four-domain structure of calmodulin appears to be retained in squid CaBP, which is consistent with the previously reported presence of four calcium-binding sites per molecule (Sheldon, A., and Head, J. F. (1988) J. Biol. Chem. 263, 14384-14389). The two tyrosines of squid CaBP are located in different halves of the molecule, one at the position corresponding to Tyr-138 in calmodulin, the other in an equivalent position in domain II. In addition, squid CaBP exhibits several differences in the region corresponding to the long central helix of calmodulin. These differences include the replacement of Lys-77 by glycine, Asp-78 by proline, and Ser-81 by proline. The sequence of this portion of the squid CaBP molecule suggests the protein is unlikely to possess the continuous long central helix found in calmodulin.

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.

Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II).

Biochem Cell Biol. 1989; 67: 179-86

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The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.

Comparison of the enhanced steady-state diffusion of calcium by calbindin-D9K and calmodulin: possible importance in intestinal calcium absorption.

Cell Calcium. 1989; 10: 189-203

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The diffusion of calcium was measured using the unidirectional flux of 45Ca across an aqueous layer. The aqueous layer was bounded by two dialysis membranes and convection was eliminated by gelling the aqueous layer with agarose. The apparent self-diffusion coefficient was determined by the dependence of the tracer flux on the diffusion distance. The apparent self-diffusion coefficient increased linearly with the concentration of calbindin-D9K and calmodulin, but the effect of calmodulin was markedly less than that of calbindin-D9K. This difference is attributed to the lower association constant for calmodulin. The ion-exchange resin Chelex-100 also increased the steady-state of 45Ca, but the effect of Chelex-100 was much less efficient than the effect of calbindin-D9K. The mechanism of enhanced diffusion was attributed to an enhanced gradient of total 45Ca. These results indicate that the steady-state unidirectional calcium flux is a superposition of free calcium diffusion and bound calcium diffusion, with only a small contribution due to a 'bucket brigade' mechanism. We suggest that this phenomenon may be important in calcium absorption across the intestine.

A technique for proton labelling of selected amino acids in deuterated calbindin D9K, heterologously expressed in E. coli, was developed in order to simplify and obtain higher resolution in 1H-NMR spectra. The spectra from two double-labelling experiments, Val plus Ser and Val plus Leu, when compared to the uniformly protonated protein showed a dramatically simpler pattern with low background signals and gave considerably sharper resonances due to reduced relaxation rates in the deuterated proteins. The selective proton labelling technique will enable detailed and rapid analysis of interesting domains of proteins and will also make the analysis of larger proteins feasible.

Refined structure of baboon alpha-lactalbumin at 1.7 A resolution. Comparison with C-type lysozyme.

J Mol Biol. 1989; 208: 99-127

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The solution of the structure of alpha-lactalbumin from baboon milk (Papio cynocephalus) at 4.5 A resolution using the isomorphous replacement method has been reported previously. Initial refinement on the basis of these low-resolution studies was not successful because of the poor isomorphism of the best heavy-atom derivative. Because of the striking similarity between the structure of lysozyme and alpha-lactalbumin, a more cautious molecular replacement approach was tried to refine the model. Using hen egg-white lysozyme as the starting model, preliminary refinement was performed using heavily constrained least-squares minimization in reciprocal space. The model was further refined using stereochemical restraints at 1.7 A resolution to a conventional crystallographic residual of 0.22 for 1141 protein atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.015 A, and for angle distances it is 0.027 A. The refinement was carried out using the human alpha-lactalbumin sequence and "omit maps" calculated during the course of refinement indicated eight possible sequence changes in the baboon alpha-lactalbumin X-ray sequence. During the refinement, a tightly bound calcium ion and 150 water molecules, of which four are internal, have been located. Some of the water molecules were modelled for disordered side-chains. The co-ordination around the calcium is a slightly distorted pentagonal bipyramid. The Ca-O distances vary from 2.2 A to 2.6 A, representing a tight calcium-binding loop in the structure. The calcium-binding fold only superficially resembles the "EF-hand" and presumably has no evolutionary relationship with other EF-hand structures. The overall structure of alpha-lactalbumin is very similar to that of lysozyme. All large deviations occur in the loops where all sequence deletions and insertions are found. The C terminus appears to be rather flexible in alpha-lactalbumin compared to lysozyme. The experimental evidence supports the earlier predictions for the alpha-lactalbumin structure that were based upon the assumption that alpha-lactalbumin and lysozyme have similar three-dimensional structures, with minimal deletions and insertions. A detailed comparison of the two structures shows striking features as well as throwing some light on the evolution of these two proteins from a common precursor.

We have purified three 35-kDa calcium- and phospholipid-binding proteins from rat liver. These three calcimedins bind to phosphatidylserine in a calcium-dependent manner and have been termed 35 alpha, 35 beta, and 35 gamma based on their relative charge as determined by isoelectric focusing. Purification of the three 35-kDa calcimedins is achieved by phenyl-Sepharose, ion exchange, and gel filtration chromatography. Antibody was produced against the annexin consensus peptide, Lys-Ala-Met-Lys-Gly-Leu-Gly-Thr-Asp-Glu, which was derived from the sequence of several Ca2+/phospholipid-binding proteins including calpactin, lipocortin, endonexin II, 67-kDa calelectrin, lymphocyte 68-kDa protein, and protein II. Recognition of each 35-kDa calcimedin by anticonsensus sequence antibody places them in this protein family. Antibodies against each 35-kDa calcimedin were raised and purified by antigen-affinity chromatography. Each antibody is monospecific for the respective 35-kDa calcimedin. Immunological cross-reactivity defines 35 alpha, 35 beta, and 35 gamma as lipocortins III, IV, and V, respectively. Surveys by immunoblot analysis using these monospecific antibodies demonstrate a markedly different tissue expression pattern for each 35-kDa calcimedin. Furthermore, the levels of 35 alpha, 35 beta, and 35 gamma are differentially regulated in maturing rat ovary and uterus. Each calcimedin has been localized by indirect immunofluorescence within specific cell types. These results support the concept that mediation of the intracellular calcium signal can occur via multiple pathways through several related yet independent mediator proteins.

Calpain and calpastatin are known to be very widely distributed in animal cells. Although the physiological roles of calpain have not yet been clarified, the proteinase-proteinase inhibitor system has been suggested to play important roles in various cellular functions coupled with Ca2+ mobilization. By nucleotide sequencing of the cloned cDNAs, the primary structures of calpains and calpastatins have recently been disclosed. The expression of the genes for calpains and calpastatins in vitro as well as in vivo is being studied using cDNAs and their fragments. This paper reviews several topics on the recent progress of calpain research.

1H NMR sequential resonance assignments, secondary structure, and global fold in solution of the major (trans-Pro43) form of bovine calbindin D9k.

Biochemistry. 1989; 28: 7065-74

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A wide range of two-dimensional 1H NMR experiments have been used to completely assign the 500-MHz 1H NMR spectrum of recombinant Ca2+-saturated bovine calbindin D9k (76 amino acids, Mr = 8500). In solution, calbindin D9k exists as an equilibrium mixture of isoforms with trans (75%) and cis (25%) isomers of the peptide bond at Pro43 [Chazin et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2195-2198], which results in two sets of 1H NMR signals from approximately half of the amino acids. The complete 1H NMR assignments for the major, trans-Pro43 isoform are presented here. By use of an integrated strategy for spin system identification, 62 of the 76 spin systems could be assigned to the appropriate residue type. Sequence-specific assignments were then obtained by the standard method. Secondary structure elements were identified on the basis of networks of sequential and medium-range nuclear Overhauser effects (NOEs), 3JHN alpha spin coupling constants, and the location of slowly exchanging amide protons. Four helical segments and a short beta-sheet between the two calcium binding loops are found. These elements of secondary structure and a few additional long-range NOEs provide the global fold. Good agreement is found between the solution and crystal structures of the minor A form of bovine calbindin D9k and between the solution structures of the minor A form of bovine calbindin D9k and intact porcine calbindin D9k.

Modulation of ATPase activities in the central nervous system by the S-100 proteins.

Neurochem Res. 1989; 14: 761-4

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The isomeric forms of bovine S-100a and S-100b have been shown to stimulate ATPase activities in fractions enriched in myelin and mitochondria isolated from the Gerbil brain and for S-100b more effectively than for calmodulin in erythrocytes or skeletal muscle. In the presence of Ca2+, S-100a produced a slight increase of ATPase activity in the mitochondrial fraction. However, S-100b in the presence of Zn2+ almost doubled the ATPase activity in brain myelin. S-100a, or S-100b, with or without Ca2+ and Zn2+ respectively, had no effect on the ATPase activity in mitochondria of the Gerbil liver. The observations may indicate a "second messenger" role for S-100b in the presence of Zn2+ in the Schwann cell.

A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation.

Biochem J. 1989; 263: 929-35

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A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.

Calbindin-D9k, a vitamin D-dependent Ca2+-binding protein, is closely associated with the transcellular absorption of calcium by mammalian enterocytes. Studies were performed to determine whether physiological concentrations of calbindin-D9k altered Ca2+ transport by the ATP-dependent Ca2+ pump in rat duodenal basolateral membrane vesicles. In solutions where free Ca2+ was buffered by EGTA, only a small stimulation of Ca2+ uptake rates could be demonstrated, and it was likely that this was secondary to changes in free Ca2+ concentration. However, a threefold stimulation of uptake by 30 microM calbindin-D9k was found when EGTA-free solutions were used, and changes in free Ca2+ activity or 45Ca2+ specific activity were avoided. The affinity for Ca2+ was reduced in this system but appeared to be stimulated by either calbindin-D9k or EGTA. Other Ca2+-binding proteins that bind Ca2+ in the micromolar range were found to increase Ca2+ uptake in the absence of EGTA. These experiments suggest that one of the actions of calbindin-D9k is to stimulate the rate of extrusion of Ca2+ from the enterocyte by increasing Ca2+ transport by the Ca2+ pump.

Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding.

J Biol Chem. 1989; 264: 12794-9

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Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.

Seven years have elapsed since the terms calpain and calpastatin were introduced. During these years, significant progress in research has been recorded. Thus, cloning and sequencing of cDNAs for calpains I and II and calpastatin have established amino acid sequences of these molecules. Structure-function relationship of calpastatin has been studied using mutated cDNAs expressed in E. coli. Interleukin 2 receptor-linked expression of calpastatin in HTLV-I-infected T-cells has been reported. Evidence for Ca2+-induced translocation of calpain to the cell membrane, followed by its autolytic activation, has been discussed. A great varieties of proteins such as several kinases, membrane and cytoskeletal proteins, and hormone receptors have been reported to be susceptible to calpains. This paper is to summarize our current knowledge on chemistry and biology of calpain and calpastatin and thereby to speculate the true function of calpains and their regulatory mechanisms.

Carp parvalbumin coordinates calcium through one carbonyl oxygen atom and the oxygen-containing side chains of 5 amino acid residues, or 4 residues and a water molecule, in a helix-loop-helix structural motif. Other calcium-binding proteins, including calmodulin and troponin C, also possess this unique calcium-binding design, which is designated EF-hand or calmodulin fold. Parvalbumin has two such sites, labeled CD and EF. Each of the calcium-binding sites of refined structures of proteins belonging to this group has a 7-oxygen coordination sphere except those of the structure of parvalbumin as it was reported in 1975. This structure had been refined at 1.9 A using difference Fourier techniques on film data. The CD site appeared to be 6-coordinate and the EF site 8-coordinate. Results of NMR experiments using 113Cd-substituted parvalbumin, however, indicate that the sites are similar to one another with coordination number greater than 6. To resolve the inconsistency between crystallographic and NMR results, 1.6 A area detector data was collected for native and cadmium-substituted parvalbumin; the structures have been refined to R factors of 18.7% and 16.4%, respectively, with acceptable geometry and low errors in atomic coordinates. Differences between the parvalbumin structure described in 1975 and the present structure are addressed, including the discovery of 7-coordination for both the CD and EF sites.

The 1H nuclear magnetic resonance (NMR) spectrum of Ca2+-saturated porcine calbindin D9k (78 amino acids, Mr 8800) has been assigned. Greater than 98% of the 1H resonances, including spin systems for each amino acid residue, have been identified by using an approach that integrates data from a wide range of two-dimensional scalar correlated NMR experiments [Chazin, Rance, & Wright (1988) J. Mol. Biol. 202, 603-626]. Due to the limited quantity of sample and conformational heterogeneity of the protein, two-dimensional nuclear Overhauser effect (NOE) experiments also played an essential role in the identification of spin systems. On the basis of the pattern of scalar connectivities, 43 of the 78 spin systems could be directly assigned to the appropriate residue type. This provided an ample basis for obtaining the sequence-specific resonance assignments. The elements of secondary structure are identified from sequential and medium-range NOEs, values of 3JNH alpha, and the location of slowly exchanging backbone amide protons. Four well-defined helices and a mini beta-sheet between the two calcium binding loops are present in solution. These elements of secondary structure and a few key long-range NOEs provided sufficient information to define the global fold of the protein in solution. Generally good agreement is found between the crystal structure of the minor A form of bovine calbindin D9k and the solution structure of intact porcine calbindin D9k. The only significant difference is a short one-turn helix in the loop between helices II and III in the bovine crystal structure, which is clearly absent in the porcine solution structure.

A Ca2+-binding protein was purified from mouse Ehrlich ascites-tumour cells. The protein forms monomers and disulphide-linked dimers, which can be separated by reverse-phase h.p.l.c. A partial amino acid sequence analysis demonstrated that the protein has an EF-hand structure. A striking homology was found to rat and human calcyclin (a member of the S-100 protein family), which is possibly involved in cell-cycle regulation.

Cloning and sequencing of a calcium-binding protein regulated by cyclic AMP in the thyroid.

EMBO J. 1989; 8: 111-6

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p24 is a thyroid protein (Mr 24,000) identified by two-dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up-regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target-site for phosphorylation by cyclic AMP-dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through 'EF hand' domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+-phosphatidylinositol cascades.

Conservation of residue interactions in a family of Ca-binding proteins.

Protein Eng. 1989; 2: 589-96

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In the TNC family of Ca-binding proteins (calmodulin, parvalbumin, intestinal calcium binding protein and troponin C) approximately 70 well-conserved amino acid sequences and six crystal structures are known. We find a clear correlation between residue contacts in the structures and residue conservation in the sequences: residues with strong sidechain-sidechain contacts in the three-dimenesional structure tend to be the more conserved in the sequence. This is one way to quantify the intuitive notion of the importance of sidechain interactions for maintaining protein three-dimensional structure in evolution and may usefully be taken into account in planning point mutations in protein engineering.

Antibodies generated against synthetic peptides that correspond to highly conserved sequence motifs in the annexins reacted with a variety of annexins from different species. These include Xenopus laevis and Drosophila melanogaster, which contain cross-reacting polypeptides of apparent Mr 34,000 and 30,000. As expected for typical annexins, the two Drosophila proteins interact in a Ca2+-dependent manner with phosphatidylserine liposomes.

A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.

The calcium-binding site in the galactose chemoreceptor protein. Crystallographic and metal-binding studies.

J Biol Chem. 1989; 264: 20817-21

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We have determined the relative affinities in solution for various metals which bind to the lone calcium-binding site of the D-galactose-binding protein which resembles the EF-hand loop. In order of affinity the metals are: Ca2+ approximately Tb3+ approximately Pb2+ greater than Cd2+ greater than Sr2+ greater than Mg2+ much greater than Ba2+. The binding affinity for calcium (Kd = 2 microM) and the slow off-rate determined for terbium (1 x 10(-3) s-1) and that the metal-binding site is unperturbed by sugar binding argue for a structural role. Furthermore, we have crystallographically refined the structure of the binding protein with the calcium substituted by cadmium, compared it with the calcium-bound structure, and found them to be identical. The results of these structural and solution studies support the hypothesis that for a given metal-binding loop, cation hydration energy, size, and charge are major factors contributing to binding affinity.

Identification of phospholipid-dependent calcium-binding proteins as constituents of matrix vesicles.

J Biol Chem. 1989; 264: 10917-21

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Uptake of mineral ions by isolated matrix vesicles (MV) incubated in synthetic cartilage lymph follows a consistent pattern. After an initial lag period, MV rapidly accumulate large amounts of Ca2+ and Pi before the appearance of crystalline mineral. The ability of MV to accumulate Ca2+ is readily destroyed by proteases, indicating that proteins are important in Ca2+ accumulation. Since MV contain significant amounts of phosphatidylserine (PS), an acidic phospholipid with affinity for Ca2+, it seemed probable that this lipid might also contribute to Ca2+ binding. The development of methods for reproducible isolation of pure active MV enabled us to search for factors responsible for the rapid accumulation of Ca2+. Reported here are studies which reveal that a set of intensely staining MV proteins, extractable with EGTA, selectively bind to Ca2+, but only in the presence of acidic phospholipids. These 30-36-kDa proteins form readily sedimentable insoluble ternary complexes of protein, Ca2+, and lipid in the presence of low levels of Ca2+. With liposomes composed of PS, alone or in combination with phosphatidylethanolamine, submicromolar levels of Ca2+ or certain other divalent cations, but not Mg2+, are sufficient to form the complexes. The physical and chemical properties of these MV proteins appear to be like those of the calpactin family of membrane-associated proteins. In fact, these MV proteins were found to cross-react with antibodies to calpactin II. Thus, calpactins appear to be important protein constituents of avian growth plate MV. This finding helps explain the enrichment in PS previously noted in MV and may also point to the mechanism by which MV rapidly accumulate Ca2+.

On the ion selectivity in Ca-binding proteins: the cyclo(-L-Pro-Gly-)3 peptide as a model.

Proc Natl Acad Sci U S A. 1989; 86: 7880-4

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Calcium plays a crucial role in many cellular processes. Its functions are directly dependent on the high specificity for Ca2+ exhibited by the proteins and ion carriers that bind divalent ions. To elucidate the basis for this specificity we have calculated the relative energies of solvation of calcium and magnesium ions in complexes with cyclo(-L-Pro-Gly-)3, a small synthetic peptide that binds Ca2+ with an affinity comparable to those of the naturally occurring proteins. The results show that the ion selectivity of the peptide resides in the difference in the solvation energies of the competing ions in water. Although the peptide is able to complex Mg2+ better than Ca2+ in the stoichiometries in which cyclo(-L-Pro-Gly-)3 binds divalent ions, it is not always able to provide as much stabilization for Mg2+ as water does. These results also explain why cyclo(-L-Pro-Gly-)3 binds Ca2+ and Mg2+ with different stoichiometries and indicate the source for expected differences in the structures of complexes of the two ions.

Two associated calcium-binding proteins (CaBPs) have recently been identified specifically in cells of myeloid origin. These proteins have relative molecular masses (Mr) of 8,000 and 14,000 and are variously referred to as the cystic fibrosis antigen, the L1 light chain, MRP-8 or p8, and the L1 heavy chain, MRP14 or p14, respectively. The expression of p8 and p14 seems to be confined to a specific stage of myeloid cell differentiation, because both proteins are expressed in circulating neutrophils and monocytes but not in normal tissue macrophages. In chronic inflammatory conditions, however, such as rheumatoid arthritis, macrophages in affected tissues express both p8 and p14. These proteins are members of a family of CaBPs of low Mr, which include S-100 alpha and beta proteins, calcyclin (2A9), intestinal CaBP and p11. All the proteins have an Mr of approximately 10,000 with the exception of p14 which has a longer C-terminal sequence after the second calcium-binding domain. Little is known about their function, although by analogy with calmodulin they could be molecules involved in intracellular signalling that are activated by an increase in the intracellular Ca2+ concentration ([Ca2+]). Here we report that p14 is phosphorylated in both monocytes and neutrophils. The level of p14 phosphorylation can be increased by elevating the [Ca2+]i using the ionophore ionomycin, but is not affected by activation of protein kinase C using phorbol 12,13-dibutyrate. The phosphorylated residue is threonine at position 113, which is the penultimate amino acid in p14 and contained in the longer 'tail' sequence. Part of this sequence is identical to the neutrophil immobilizing factors NIF-1 and NIF-2, indicating that the phosphorylation event could have a role in the generation of NIF activity in the p14 protein.

The non-lysosomal, calcium-dependent proteolytic system of mammalian cells.

Revis Biol Celular. 1989; 20: 139-59

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The intracellular calcium-dependent proteases (calpains) and their endogenous protein inhibitor (calpastatin) are present in many different mammalian cells. There is emerging evidence for their importance in the turnover of membrane-associated proteins. Accordingly, it is important to understand how these proteinases and their inhibitor interact within cells, in particular at membranes. Bovine myocardial calpastatin appears to be associated in part with intracellular membranes, where it may effectively block the activity of calpain II on membrane-associated proteins. Immuno-electron microscopic studies suggest that canine myocardial calpain and calpastatin are associated with a number of membranous organelles. During canine myocardial autolysis, the amount of calpain at various organelles decreased, but the amount of calpastatin decreased to an even greater extent. Thus there may be a high calpain to calpastatin balance during heart ischemia at these sites. Calpain II aggregation may contribute to localization of the proteinase at sites of high calcium concentration within cells. A model is presented for interaction of calpain II and calpastatin at cellular membranes in the presence of calcium.

Characterizations of two distinct Ca2+-dependent phospholipid-binding proteins of 68-kDa isolated from human placenta.

J Biol Chem. 1989; 264: 17222-30

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Two distinct 68-kDa proteins, named 68K-I (pI 6.4) and 68K-II (pI 5.6), were solubilized from human placenta by treatment with 5 mM EGTA. On DE52 cellulose column chromatography at pH 7.4, 68K-I in the EGTA eluate was recovered in the unadsorbed fractions, whereas 68K-II was retained on the column and eluted with 0.2 M NaCl. The 68K-I protein was obtained in more than 95% purity by further hydroxylapatite and cation exchange chromatographies, while the 68K-II protein was purified further by gel filtration and hydroxylapatite chromatographies. Partial amino acid sequence data showed that 68K-I protein was a novel protein which shared the same sequences as lipocortin I and that 68K-II was the same as human p68/67-kDa calelectrin (Crompton, M. R., Owens, R. J., Totty, N. F., Moss, S. E., Waterfield, M.D., and Crumpton, M. J. (1988) EMBO J. 7, 21-27; Sudhof, T. C., Slaughter, C. A., Leznicki, I., Barjon, P., and Reynolds, G. A. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 664-668). The two proteins bound to acidic phospholipids, phosphatidylserine, and/or phosphatidylinositol, but not to phosphatidylcholine, in the presence of micromolar levels of Ca2+. 68K-I bound to phosphatidylinositol preferentially to phosphatidylserine, whereas 68K-II bound only to phosphatidylserine. Both 68K-I and 68K-II inhibited phospholipase A2 activity, and the inhibition by 68K-II was detectable only in the presence of 100 mM KCl. 68K-I, but not 68K-II, was found to bind to F-actin in a Ca2+-dependent (1 mM) manner. Moreover 68K-I, but not 68K-II, was phosphorylated in vitro at tyrosine residues by fps kinase and by epidermal growth factor receptor/kinase, the latter reaction being dependent on Ca2+ and epidermal growth factor. Western blot analysis with affinity purified anti-68K-I and anti-68K-II antibodies showed that 68K-I was located in only certain tissues, especially human placenta, whereas 68K-II was present in many human and rat tissues.

Calbindin-D9k was quantified and its cellular location was defined in uterus, yolk sac, and placenta. In late gestation (days 17 to term) coordinated induction of calbindin-D9k was seen in uterine epithelial lining cells and juxtaposed yolk sac visceral epithelium as well as the intraplacental yolk sac epithelium. The induction of calbindin-D9k in these cells coincided with the time of exponential fetal bone growth and maximal fetal accumulation of calcium, suggesting a role of the protein in these epithelial layers in maternal-fetal calcium transport. Dynamic changes also occurred in the calbindin-D9k contents of the two layers of uterine smooth muscle (outer longitudinal and inner circular) during mid- and late gestation. During early pregnancy (days 0-4), calbindin-D9k was present in the two smooth muscle layers. By midgestation (day 10), calbindin-D9k had decreased by a factor of 10 in these tissue layers. During late gestation calbindin-D9k rebounded in the inner circular smooth muscle layer. These uterine changes of early and midgestation were reproduced by the endocrine changes of pseudopregnancy. Progesterone appeared to be a good candidate for controlling the midgestational decrease of uterine muscle calbindin-D9k, as it blunted estrogen's induction of the protein in the muscle layers and stroma in a dose-dependent manner. Changes in myometrial calbindin-D9k may reflect variations in muscular calcium storage, thereby representing alterations in potential for contraction.

Characterization and site-specific mutagenesis of the calcium-binding protein oncomodulin produced by recombinant bacteria.

J Biol Chem. 1989; 264: 3470-7

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A bacterial expression system for the parvalbumin-like calcium-binding protein oncomodulin has been constructed. This system can yield 50-fold more oncomodulin than the richest known mammalian source, the rat Morris hepatoma 5123. The bacterially produced protein folded correctly as monitored by UV, fluorescence, and 1H nuclear magnetic resonance spectroscopy and is immunologically identical to rat hepatoma oncomodulin. A calcium-specific conformational change is observed in the bacterial oncomodulin similar to that of the hepatoma protein. A modification of the putative calcium-specific CD loop by site-directed mutagenesis, which changed Asp-59 to Glu-59, eliminates calcium-specific effects. In contrast to the native molecule, the mutant Glu-59 now exhibits a magnesium-induced conformational change when monitored by UV difference or fluorescence excitation spectroscopy. The availability of large amounts of bacterially produced oncomodulin combined with the ability to modify at will the metal-binding ligands should now allow dissection of the unusual pairing in oncomodulin of one calcium-specific calmodulin-like site with one calcium/magnesium parvalbumin-like site.

The amino acid sequence of D2 protein was compared with those of calcium binding proteins and receptor for calcium channel blockers in connection with the data showing the participation of Ca2+ in photosystem 2 electron transport and the inhibition of this process by calmodulin antagonists, calcium channel blockers and local anesthetics. Protein D2 possesses a pattern analogous to the "EF-hand" sites of the calcium binding proteins. Comparison of the amino acid sequence of the calmodulin fragment binding the phenothiazine type calmodulin antagonists with the amino acid sequence of D2 protein and calcium channel protein revealed a high degree of sequence identity. Common structural features take place also between the membrane spanning segment III of D2 protein, which contains the tyrosine residue (161), responsible for ESR-signal IIS, and the membrane segment IVS5 of calcium channel protein. A model explaining the mechanism of calcium function in the oxygen-evolving system is proposed.

A 24-kDa protein of Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, is recognized by antisera from both humans and experimental animals infected with this organism. Near its C terminus are two regions that have sequence similarity with several Ca2+-binding proteins and that conform to the "E-F hand" Ca2+-binding structure. We expressed a cDNA encoding this protein in Escherichia coli and showed that both the recombinant protein and the 24-kDa native trypanosome protein do indeed bind Ca2+. The protein's low Ca2+-binding capacity (less than 2 mol of Ca2+/mol of protein) and high Ca2+-binding affinity (apparent Kd less than 50 microM Ca2+) are consistent with binding of Ca2+ via the E-F hand structures. Immunofluorescence assays using a mouse antiserum directed against the fusion protein localized the native protein to the trypanosome's flagellum. The protein's abundance, Ca2+-binding property, and flagellar localization suggest that it participates in molecular processes associated with the high motility of the parasite.

The cytoskeleton-associated protein vinculin exhibits the shape of a 'balloon on a string' when examined by rotary shadowing electron microscopy. Recently, the complete primary structure of chicken vinculin was determined which leads to the questions as to whether the globule might be either hollow or disc shaped, or whether the electron micrographs resemble those of vinculin dimers. Based on a hydrodynamical theory, evidence is presented for the assumption that vinculin as a monomer consists of a compact spherical head 6 nm in diameter connected by a proline-rich domain to a rod-shaped tail about 20 nm in length.

Identification of a cell cycle-dependent gene product as a sialic acid-binding protein.

Biochem Biophys Res Commun. 1989; 163: 506-12

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A Ca2+-dependent sialic acid-binding protein was purified on fetuin-Sepharose from various types of human tissue. The molecular mass was determined to be 10,315 Da by laser desorption mass spectrometry. Partial sequence analysis after cyanogen bromide cleavage that yielded one N-terminus accessible for Edman degradation revealed an identity to an internal stretch following the only methionine residue within a putative amino acid sequence (Mr 10,048), deduced from the cDNA of a cell cycle-specific gene. The reported biochemical identification is a prerequisite to infer the biological role of the so far undetected gene product. Initial glycohistochemical studies with sialic acid-(BSA-biotin) raised evidence for nuclear localization of sialic acid-binding sites that might reflect, at least in part, detection of this protein.

Approaches to study the role of S100 proteins in calcium-dependent cellular responses.

J Dairy Sci. 1988; 71: 2028-34

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Calcium is necessary for the optimal growth and cellular functions of most living organisms. For example, Ca is involved in the processes of muscle contraction, stimulus-secretion coupling, bone formation, blood clotting, cell proliferation and motility, and fertilization. Many regulatory actions of Ca are mediated through Ca-binding proteins. Calcium-modulated proteins are a subclass of Ca-binding proteins that are thought to be the major signal transducers of Ca acting as a cellular second messenger. Most Ca-modulated proteins are not enzymes but are effector proteins capable of transducing a Ca signal into a biological response by their ability to bind Ca reversibly and modulate the activity of other proteins in a Ca-dependent manner. This review focuses on a set of Ca-modulated proteins, the S100 proteins, and their possible roles in mediating Ca-dependent cellular events.

Carboxyl-terminal truncation and site-directed mutagenesis of the EF hand structure-domain of the small subunit of rabbit calcium-dependent protease.

J Biochem (Tokyo). 1988; 104: 927-33

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A mutant of the small subunit of rabbit calcium-dependent protease lacking the amino-terminal one-fourth produced in Escherichia coli could associate with the native large subunit to exert protease activity. Deletion of a few carboxyl-terminal residues of this variant small subunit caused a significant decrease in the protease activity after reconstitution with the native large subunit. Loss of the fourth EF hand loop region by further truncation of the variant small subunit made interaction with the large subunit impossible. The calcium binding assay revealed that the fourth EF hand structure of the rabbit small subunit, which has been previously demonstrated to possess two calcium-binding sites, can bind calcium ions. Furthermore it was established by site-directed mutagenesis that the first EF hand structure, in addition to the fourth one, is capable of binding calcium ions. Replacement of amino acids in the EF hand structure affected interaction with the native large subunit or the calcium sensitivity of the reconstituted product. These findings indicate that the EF hand structure-domain of the small subunit is essential for the full protease activity.

Synthesis and expression of a gene coding for the calcium-modulated protein S100 beta and designed for cassette-based, site-directed mutagenesis.

J Biol Chem. 1988; 263: 7830-7

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As an initial step in studies aimed at addressing the question of what common and unique features of the S100 family of proteins are related to their specific functions and localizations, a gene coding for one of the S100 proteins, S100 beta, has been prepared by ligation of 12 overlapping, synthetic oligonucleotides. Automated DNA sequence analysis demonstrated that the final construct has the expected structure. The gene was inserted into a plasmid vector that contains a tac promoter and ampicillin-resistance gene, thus allowing both amplification and direct expression cloning in Escherichia coli. The gene was designed to allow rapid, efficient changes of single or multiple amino acids by using cassette-based mutagenesis while the gene is resident in the vector. The expressed protein (VUSB-1) is indistinguishable from bovine brain S100 beta in terms of electrophoretic mobility, reactivity with antibodies to S100 beta, amino acid composition, and partial amino acid sequence analysis. Preparations of expressed protein are also functionally similar to bovine brain S100 beta as determined by aldolase activator activity and neurite extension factor activity, supporting the concept that these activities are a property of the S100 beta polypeptide.

Fluorimetric titrations of parvalbumin II (pI 4.2) of pike (Pike II) with Ca2+ and Tb3+ show the CD and EF binding sites to be non-equivalent. The intrinsic binding constants of the strong and the weak sites obtained for Ca2+ are: KsCa = 1.6.10(8) M-1; KwCa = 6.6.10(5) M-1. Differences of the order of 100% were encountered between the Tb3+ binding constants obtained with four different versions of titration. Their average values are: KsTb = 1.9.10(11) M-1; KwTb = 1.0.10(7) M-1. The distances of the strong and the weak sites from the singular Tyr-48, rs = 9.5 A and r2 = 11.5 A, were derived from Forster-type energy transfer and proved compatible with the X-ray structure of parvalbumin III (pI 4.2) of carp (CarpIII). From the distances, it is suggested that CD is the strong and EF the weak metal-binding site of PikeII. Tb3+ was shown by CD spectroscopy to have the same structural effect on PikeII as Ca2+. Removal of the metal ions from PikeII results in a decrease of helix content as monitored by CD spectroscopy. This decrease is larger than that in CarpIII. A concomitant decrease of the fluorescence quantum yield at nearly constant decay time is indicative of mainly static quenching, probably by the non-coordinating carboxylate groups. The maximum helix content is almost completely reestablished upon binding of the first metal ion. However, small changes of the energy transfer in PikeII with one terbium ion bound to the strong site indicate fine structural rearrangements of the strong binding site when Ca2+ is bound to the weak one.

The kinetics of calcium dissociation from wild-type bovine calbindin D9k (the smallest protein known with a pair of EF-hand calcium-binding sites) and five mutants with single amino-acid substitutions and/or deletions has been studied by stopped-flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N-terminal half of the molecule, at or near the first calcium-binding site (I). Substitutions and deletions of amino acids in the calcium-binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C-terminal calcium-binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca-NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr-13 and Glu-35, an interaction linking the two alpha-helices flanking site I, through replacement of Tyr-13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein. Comparison of this kinetic data with binding-constant data, previously obtained in our laboratories, shows that the decrease in Ca2+-affinity of site I, observed in most mutants, is predominantly due to an increased off-rate from this site. At low ionic strengths the second-order rate constants for Ca2+-binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 10(9) M-1 s-1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature.

Spasmin-like proteins in various ciliates revealed by antibody to purified spasmins of Carchesium polypinum.

Experientia. 1988; 44: 768-71

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It was found that some ciliates, Stentor, Spirostomum and Blepharisma, which can contract rapidly like the stalks of Vorticellidae, have Ca2+-binding proteins that are very similar to spasmins, in the immunological sense. The presence of spasmins in other Protozoa and in some Metazoa was also investigated.

The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this superfamily. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.

Calcium binding to whiting parvalbumin induces large changes in the fluorescence, absorption, and circular dichroism spectra of the protein. The fluorescence emission maximum of the single tryptophan shifts from 325 to 348 nm upon the removal of calcium and decreases in intensity by 50%. All of the spectral changes are linear between 0 and 2 mol of calcium bound/mol of protein, which suggests that the only protein species present in significant concentration are PA0 and Pa-Ca2. The kinetics of calcium binding measured by stopped-flow fluorescence are accurately single exponential from 2 X 10(-7) to 2 X 10(-4) M free calcium. The kinetics of calcium dissociation show a pronounced lag and are best fit by two rate constants of 1.2 and 3.0 s-1. The minimal kinetic mechanism that adequately describes the rate and equilibrium data is a branched pathway mechanism in which the rate and equilibrium constants are markedly different for each pathway: (formula; see text) At [Ca] less than 2 microM the upper kinetic pathway of calcium binding predominates whereas at [Ca] greater than 2 microM calcium binding occurs predominantly by the lower kinetic pathway. Calcium dissociates primarily by the upper kinetic pathway.

Amino acid sequence of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor.

J Biol Chem. 1988; 263: 15378-85

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Muscles of invertebrate species contain abundant quantities of soluble, sarcoplasmic, high affinity Ca2+-binding proteins (SCBPs). The SCBPs belong to the calmodulin superfamily and contain four homologous domains (I-IV) which arose by reduplication of a gene for a small ancestral protein. We have determined the amino acid sequence of the SCBP from the sandworm Nereis diversicolor. This protein is the only SCBP which has been crystallized in a form suitable for three-dimensional structure determination by high-resolution x-ray analysis (Babu, Y. S., Cox, J. A., and Cook, W. J. (1987) J. Biol. Chem. 262, 11184-11185). N. diversicolor SCBP is a single polypeptide chain of 174 amino acids, including single residues of glutamine and histidine, 2 tyrosines, and 3 tryptophans. It is devoid of cysteine and has an acetylated amino terminus, a calculated Mr of 19,485, and a net charge of -13 at neutral pH. There was no evidence for heterogeneity in the sequence. Probable Ca2+-binding sites were recognized in domains I, III, and IV. Comparison with other available invertebrate SCBP sequences shows an unusually high degree of variability among these proteins, with only 9 residues common to all species.

Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation.

Mol Cell Biol. 1988; 8: 2402-10

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The cellular mechanisms involved in chronic inflammatory processes are poorly understood. This is especially true for the role of macrophages, which figure prominently in the inflammatory response. Two proteins, MRP8 and MRP14, which are expressed in infiltrate macrophages during inflammatory reactions but not in normal tissue macrophages, have been characterized. Here we report that MRP8 and MRP14 mRNAs are specifically expressed in human cells of myeloid origin and that their expression is regulated during monocyte-macrophage and granulocyte differentiation. To initiate the analysis of cis-acting elements governing the tissue-specific expression of the MRP genes, we cloned the human genes encoding MRP8 and MRP14. Both genes contain three exons, are single copy, and have a strikingly similar organization. They belong to a novel subfamily of highly homologous calcium-binding proteins which includes S100 alpha, S100 beta, intestinal calcium-binding protein, P11, and calcyclin (2A9). A transient expression assay was devised to investigate the tissue-specific regulatory elements responsible for MRP gene expression after differentiation in leukemia HL60 cells. The results of this investigation demonstrated that the cis-acting elements responsible for MRP expression are present on the cloned DNA fragment containing the MRP gene loci.

Theoretical estimation of the calcium-binding constants for proteins from the troponin C superfamily based on a secondary structure prediction method. I. Estimation procedure.

J Theor Biol. 1988; 135: 41-61

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Proteins belonging to the TNC superfamily are known to be built of two, three, four, or six domains of closely similar amino acid sequences. Each domain binds no more than one calcium ion and shows a characteristic helix-loop-helix structure when in the calcium-bound state. Conformational properties of all the domains known so far have been analysed by us using a secondary structure prediction method (Garnier, J., Osguthorpe, D.J. & Robson, B. (1978). J. molec. Biol. 120, 97). Significant differences in distribution of residues predicted as being in the helical, beta-turn, and coil conformations have been found between the strongly, weakly, and non-binding domains. We could determine the ideal prediction pattern characteristic for the domains with the highest affinity for calcium. On the basis of our analysis and observations made by other authors we worked out a few simple rules which made it possible to compare conformational properties of a given domain with the ideal reference pattern and estimate, in this way, the Ca2+-binding constant of the domain. In native proteins the domains are known to be organized in pairs. The Ca2+-binding constant for a two-domain region could be evaluated from the sum of the estimation points attributed to each of its components. Using our method it is possible to predict the binding constants of typical domains and two-domain regins with a precision of one order of magnitude. Data on amino acid sequences and calcium-binding constants of all known proteins, believed to be the members of the TNC superfamily, have been reviewed. References to virtually all papers published on this subject before the end of 1987 are given.

Conformational properties of Ca2+-binding segments of proteins from the troponin C superfamily.

Biophys Chem. 1988; 31: 133-7

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The troponin C superfamily consists of about 100 Ca2+-binding proteins. Sequence variations observed in these proteins have been analyzed and lead to the following conclusions. (1) There are some strict rules defining the set of calcium ligands necessary for effective Ca2+ binding. (2) If they are fulfilled, the Ca2+ binding constant depends on tertiary interactions within a protein, as well as the free energy of secondary structures of its polypeptide chain. The former provide a constant contribution to the free energy of protein folding and the Ca2+-binding process. (3) The observed variety in Ca2+-binding constants of these proteins results from the various abilities of segments of these proteins to assume the correct secondary structure.

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.

A discontinuous epitope on p36, the major substrate of src tyrosine-protein-kinase, brings the phosphorylation site into the neighbourhood of a consensus sequence for Ca2+/lipid-binding proteins.

FEBS Lett. 1988; 236: 201-4

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Previous models of p36 based on proteolytic fragments describe the tail and core as two noninteracting domains. However, the monoclonal antibody H28 recognizes a discontinuous epitope, which covers the peptide segments around Ser 25 in the tail and around Glu 65 in the core of porcine p36. Thus, the phosphorylatable Tyr 23 is much closer to the first consensus sequence (residues 46-62) of Ca2+/lipid-binding proteins than previously thought. This apposition is in line with biochemical experiments indicating an influence of core ligands on tyrosine phosphorylation and an enhanced Ca2+ requirement of the modified p36 in phospholipid binding.

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k.

Eur J Biochem. 1988; 175: 439-45

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The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.

S100 protein, an acidic and calcium-binding protein, was believed to be localized in the nervous tissue, but recently it has been reported to be mainly present in the cardiac and the skeletal muscles of various mammals in the alpha alpha form (S100a0) at much higher levels than the nervous tissues. We isolated here S100 protein from human cardiac muscles. The isolated cardiac muscle S100 protein showed a single band on electrophoresis at the same position as that of human skeletal muscle S100a0. The amino acid composition of the purified S100 protein was quite similar to that of human skeletal muscle S100a0 or bovine brain S100a0. The immunohistochemical study by use of antibodies monospecific to the alpha subunit of S100 protein (S100-alpha) revealed that S100-alpha was strongly labeled in human myocardial cells, whereas the beta subunit of S100 protein (S100-beta) was not detected in the cells. These results suggest that a predominant form of S100 protein in human myocardial cells is not S100a (alpha beta) or S100b (beta beta), but S100a0 (alpha alpha). In order to determine the ultrastructural localization of S100a0 in mouse cardiac muscle, the direct peroxidase-labeled antibody method was employed. S100a0 was mainly localized in the polysomes in the interfibrillar spaces, the fine filamentous structure of the Z line and fascia adherens of the intercalated disc and in the lumen of junctional sarcoplasmic reticulum.

Rabbit skeletal muscle troponin C (TnC) was investigated by means of 1H NMR in the presence of dithiothreitol that prevents dimerization of the protein. Two-dimensional (2D) 1H NMR spectra were observed in order to assign resonances to specific amino acids. One-dimensional 1H NMR spectra were observed as a function of Ca2+ concentration. The Ca2+-induced spectral change is categorized into two types: type 1 corresponds to the conformational change of the C-terminal-half domain (Ca2+ high-affinity sites) and type 2 to that of the N-terminal-half domain (Ca2+ low-affinity sites). From the 2D NMR spectra and Ca2+ titration data, it was suggested that (1) amide protons of Gly-108, Ile-110, Gly-144, and Ile-146 are hydrogen-bonded when the C-terminal-half domain binds 2 mol of Ca2+ and (2) hydrogen bonds of Gly-108, Ile-110, Gly-144, and Ile-146 are destroyed or weakened when the C-terminal-half domain releases 2 mol of Ca2+. Nuclear Overhauser enhancement difference spectra as well as the Ca2+ titration data suggested that a hydrophobic cluster is formed in the C-terminal-half domain when the C-terminal-half domain binds 2 mol of Ca2+. A hydrophobic cluster exists in the N-terminal-half domain without regard to Ca2+ binding to the N-terminal-half domain. The spectra of Tyr-10 showed both types of spectral change during the Ca2+ titration. The results suggested that Tyr-10 of apo-TnC interacts with the C-terminal-half domain.

Levels and distribution of the calcium-modulated proteins S100 and calmodulin in rat C6 glioma cells.

J Neurochem. 1988; 50: 572-9

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To understand better the mechanisms involved in the transduction of a calcium signal into an intracellular response via multiple calcium-modulated proteins, we have examined the calcium-modulated proteins, S100 and calmodulin, and their intracellular targets in rat C6 glioma cells. Subconfluent, confluent, and postconfluent C6 cells contain predominantly, if not exclusively, the S100 beta polypeptide. The level of S100 beta in C6 cells increases approximately 20-fold from subconfluency to postconfluency whereas the level of calmodulin increases only about two-fold. The subcellular distribution of S100 beta and calmodulin in mitotic cells is similar. However, the subcellular distribution of these proteins in interphase cells is different and appears to change with cell density. Gel overlay analysis demonstrated that the S100- and calmodulin-binding protein profiles are significantly different and that some of the binding proteins appear to change in intensity with cell density. These data demonstrate that S100 beta is the predominant S100 polypeptide in C6 cells and suggest that changes in S100 beta and S100 beta-binding proteins may be involved in regulating S100-mediated intracellular processes in C6 cells. Our studies also suggest that the levels of S100 and calmodulin may be differentially regulated in C6 cells.

Protein engineering is a means of probing the role of electrostatic interactions in protein functions; this elegant technique has been applied to the elucidation of electrostatic effects in enzyme catalysis. Here we show how the use of mutant proteins allows the determination of the contributions of individual charges to the free energy of ion binding to proteins. We have investigated the importance of three negatively charged side chains in the binding of Ca2+ to bovine calbindin D9K (ref.2): these are clustered around the calcium sites but are not directly involved as ligands. Each of these charges is found to contribute approximately 7 kJ mol-1 to the free energy of binding of two Ca2+ ions and to affect the cooperativity of Ca2+ binding. The influence of surface charges on ion binding to proteins may be more common than generally supposed and could have important consequences for protein function.

Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand. Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium. However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins. We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene. It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin. This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.

Grasping for calcium binding sites in sodium channels with an EF hand.

J Theor Biol. 1987; 127: 451-9

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Amino acid sequences near the carboxy terminal end of the Electrophorus electricus electric organ and rat brain sodium channel polypeptides were discovered to be putative EF hand calcium binding sites. This conclusion was made using the following criteria: the Tufty-Kretsinger and Szebenyi-Moffat EF hand tests, a computer generated analysis, the revised guidelines of Chou & Fasman, and sequence comparisons with other published EF hand calcium binding regions. These results suggest that the sodium channel may be a calcium binding protein.

A novel calcium binding site in the galactose-binding protein of bacterial transport and chemotaxis.

Nature. 1987; 327: 635-8

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The refined 1.9-A resolution structure of the periplasmic D-galactose-binding protein (GBP) reveals a calcium ion surrounded by seven ligands, all protein oxygen atoms. A nine-residue loop (amino-acid positions 134-142), which is preceded by a beta-turn and followed by a beta-strand, provides five ligands from every second residue. The last two ligands are supplied by the carboxylate group of Glu 205. The entire GBP Ca2+-binding site adopts a conformation very similar to the site in the 'helix-loop-helix' or 'EF-hand' unit commonly found in intracellular calcium-binding proteins, but without the two helices. Structural analyses have also uncovered the sugar-binding site some 30 A from the calcium and a site for interacting with the membrane-bound trg chemotactic signal transducer approximately 45 A from the calcium. Our results show that a common tight calcium binding site of ancient origin can be tethered to different secondary structures. They also provide the first demonstration of a metal-binding site in a protein which is involved in bacterial active transport and chemotaxis.

The amphiphilicity of ACP helices: a means of macromolecular interaction?

FEBS Lett. 1987; 215: 261-5

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ACP interacts with diverse proteins in an unknown way. Possibly there is a similar mode of interaction between ACP and all ACP-binding proteins, the amphiphilic helix. The hydrophobicities of helices from 4 different ACPs were compared. Hydrophobic moment plots were prepared for ACP helices and those of many EF hand calcium-binding proteins. Both groups of proteins occupied the same region of the plot.

Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis.

Nature. 1987; 330: 80-2

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The aetiology and cellular mechanism of chronic inflammatory processes are poorly understood. Macrophages act prominently in the inflammatory response and we report here that they express two calcium-binding proteins. The expression of these proteins, referred to as MRP-8 and MRP-14, is specific for cells of myeloid origin, namely granulocytes, monocytes and macrophages, and is observed in blood granulocytes and monocytes but not in normal tissue macrophages. In acutely inflamed tissues, macrophages can express MRP-14 but not MRP-8, and in chronic inflammations, such as primary chronic polyarthritis, infiltrate macrophages express both MRP-8 and MRP-14. Characterization of MRP-8 and MRP-14 could therefore be useful to the understanding of cellular processes induced in chronic inflammation.

Calregulins were purified from bovine, rabbit and chicken liver, and their structural properties were compared. Significant differences between the three calregulins include a lower Mr for chicken calregulin (57,000) than for rabbit and bovine calregulin (63,000), and the glycosylation of only bovine calregulin. Amino acid composition and peptide maps of the three calregulins were very similar. No major differences were detected in the Ca2+-binding properties of the three proteins. Zn2+-induced changes in calregulin conformation and hydrophobicity monitored by intrinsic protein fluorescence and the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulphonate were very similar, suggesting that the Zn2+-dependent increase in the hydrophobicity of bovine, rabbit and chicken calregulin was conserved. These studies more fully define what is a calregulin, demonstrate that calregulin is a relatively invariant constituent of vertebrate liver, and indicate that calregulin structure has been highly conserved in bovine, chicken and rabbit liver.

Calsequestrin, a calcium-binding protein found in the sarcoplasmic reticulum of muscle cells, was purified from rabbit and canine cardiac and skeletal muscle tissue. The amino acid compositions and amino-terminal sequences of skeletal and cardiac calsequestrin from rabbit and dog were determined. The amino acid composition of the cardiac form was very similar to the skeletal form. The amino-terminal sequence of the cardiac form was homologous to, but not identical with, the amino-terminal sequence of the skeletal form of the protein. Few species differences in the amino-terminal sequences were observed. The calcium-binding capacity of the cardiac form was half the capacity of the skeletal form although the affinities of the two forms of calsequestrin for Ca2+ were similar (Kd = 1 mM). Calcium binding to the cardiac form induced structural changes in the protein as determined by circular dichroism and intrinsic fluorescence spectroscopy. The alpha-helical content of cardiac calsequestrin increased from 3.5% to 10.9% upon binding calcium, while the intrinsic fluorescence of the protein increased 14%. Potassium ions also affected the conformation of cardiac calsequestrin.

To understand the physiological role of the calcium-binding proteins S100 alpha and S100 beta, it is necessary to determine the distribution of these proteins and detect their intracellular targets in various tissues. The distribution of immunoreactive S100 alpha and S100 beta in various rat tissues was examined by radioimmunoassay. All tissues examined contained detectable S100, but the S100 beta/S100 alpha ratio in each tissue differed. Brain, adipose, and testes contained 18- to 40-fold more S100 beta than S100 alpha; skin and liver contained approximately equivalent amounts and kidney, spleen, and heart contained 8- to 75-fold more S100 alpha than S100 beta. Analysis of S100-binding proteins by gel overlay showed that each tissue possessed its own complement of binding proteins. The S100 beta-binding profile was indistinguishable from the S100 alpha-binding profile and both of these profiles were distinct from the calmodulin-binding profile. These observations suggest that the differential distribution and quantity of the individual S100 polypeptides and their binding proteins in various tissues may be important factors in determining S100 function.

The cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-beta-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4 mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.

Calcium binding protein from porcine intestine binds to phosphatidylserine vesicles in the presence of calcium.

Biochim Biophys Acta. 1987; 897: 502-6

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Protein II, a 32K cytoskeleton-associated protein isolated from porcine intestinal epithelium, binds to vesicles composed of phosphatidylserine in the presence, but not the absence, of 10 microM Ca2+. Binding was saturable and was specifically inhibited by chelation of free Ca2+ with EGTA. Binding was also inhibited by trifluophenothiazine. Vesicles composed of dimyristoylphosphatidylcholine did not bind protein II, suggesting that interaction with phosphatidylserine was selective. These properties are consistent with a possible role for protein II in Ca-regulated cytoskeleton-cell membrane events.

The complete primary structure of the cholecalciferol-induced chicken intestinal 28-kDa calbindin-D is reported. It is a single-chain polypeptide consisting of 261 amino acid residues (computed Mr = 30,042) and is blocked at the amino terminus. Tryptic digestion of the S-pyridylethylated protein followed by HPLC peptide mapping and automated sequence determination provided the bulk of the sequence information. Subsequent chymotryptic and Staphylococcus aureus V8 protease cleavages yielded the sequences of several additional regions as well as extensive overlapping of the tryptic peptides. The primary structure shows six homologous regions of sequence based on the EF-hand concept of calcium binding, four of which are predicted to actually bind calcium. Aside from these regions, there is no overall structural identity or apparent similarity with the mammalian calbindins (9 kDa), calmodulin, or troponin C. It is predicted that the secondary structure of 28-kDa calbindin-D is significantly different from the other proteins of this class, which bind four calcium atoms.

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio-Gel P-4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metallo-protein like alpha-lactalbumin, which is a homologue of hen egg white lysozyme.

Skin Ca2+-binding protein (SCaBP) was reported to be distinct from the Ca2+-binding parvalbumin (PV), however, more recently its amino acid sequence was shown to be identical to PV. We purified a protein (Mr 12,000; pI4.5) from isolated epidermis (free of other cell layers) of adult rats and whole skin (containing no PV) of newborn rats. This protein is referred to as epidermal protein (EP-12), distinct from PV in its hydrophobicity, amino acid composition and immunological properties. Previously isolated SCaBP was shown to be a mixture of EP-12 and PV. The localization and possible functions of EP-12 and of PV in skin of adult and newborn rat are discussed.

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.

Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4). 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site. The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site. The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-[[2-[bis(carboxymethyl)-amino]- 5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline or by using a Ca2+-selective electrode. The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3. Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1). The binding entropy is strongly positive in all cases. Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants. Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4. These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4). There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.

Osteocalcin (also called 'bone Gla-protein') was detected in fossil bovid bones ranging from 12,000 years to 13 million years old and in rodent teeth 30 million years old. Both the antigenic activity and the protein-bound Gla-residues have remained intact. The protein is indistinguishable from recent bovine osteocalcin when analyzed by HPLC using ion exchange and size exclusion columns. If sufficient amounts can be extracted and an adequate purification procedure is established, this would be the first time that amino acid sequences in a protein from fossil bones may be determined. Such sequence data could offer a new approach to the phylogenetic study of extinct taxa.

Calcium binding to bone gamma-carboxyglutamic acid protein (BGB) from calf has been studied using 43Ca NMR. The temperature dependence of the 43Ca NMR signal has been used to calculate the calcium ion exchange rate, koff. The dependence of the 43Ca NMR band shape on the [Ca2+]/[BGP] ratio fits well to a chemical equilibrium model having a single Ca2+-binding site with an association constant in the range of 5 X 10(3)-1 X 10(5) M-1. The pH dependence of the 43Ca NMR line-width shows a single apparent pKa value of 5.1.

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

J Cell Biol. 1986; 103: 1635-48

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Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

An immunological comparison of several novel calcium-binding proteins.

J Biol Chem. 1986; 261: 15815-8

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Polyclonal antibodies prepared against each of the calcimedins were utilized to determine their tissue distribution. The immunological survey of rat tissues revealed that the levels of the 35-kDa calcimedin varied, while the amount of the 67-kDa calcimedin was relatively constant in the tissues examined. A new immunoreactive species, 52 kDa, was detected with the antibody to the 35-kDa calcimedin; this protein appears to be the predominant immunoreactive species in the tissues examined. Antibodies to the 35-kDa calcimedin were also used to compare many other calcium-binding proteins in order to determine immunological relationships. These comparisons demonstrate that the epidermal growth factor receptor/kinase substrate (p35), the src kinase substrate (pp36), and calregulin are immunologically unrelated to the calcimedins. However, it was found that the 67-kDa calcimedin and the p70 calelectrin are identical, as are the 35-kDa calcimedin and the p32.5 calelectrin. The calimedins are a subset of the chromobindins. In addition, the antibody to the 35-kDa calcimedin also cross-reacts with synexin, which may be related to the new 52-kDa immunoreactive protein identified.

Binding sites for calcium, lipid and p11 on p36, the substrate of retroviral tyrosine-specific protein kinases.

FEBS Lett. 1986; 198: 361-4

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Biochemical and partial sequence data reveal the two-domain structure of p36. A loose structure of some 30 residues at the amino-terminus contains the phosphorylatable tyrosine and the binding site for the p11 regulatory chain. The following p33 domain retains the lipid-binding site as well as the Ca2+ site which influences the spectral properties of the single tryptophan and one tyrosine. The combined sequence data covering about 25% of the molecule identify p36 as a unique polypeptide.

Bimane- and acrylodan-labeled S100 proteins. Role of cysteines-85 alpha and -84 beta in the conformation and calcium binding properties of S100 alpha alpha and S100b (beta beta) proteins.

Biochemistry. 1986; 25: 6934-41

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Bovine brain S100 alpha alpha, S100a (alpha beta), and S100b (beta beta) protein dimers were labeled with the sulfydryl-specific fluorescent probes monobromo(trimethylammonio)bimane (bimane) and 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) at cysteines-85 alpha and -84 beta. The conformation and fluorescence properties of the S100 proteins derived were studied by means of anion-exchange chromatography on a Mono Q column using a fast protein chromatography system and fluorescence intensity, maximum emission wavelength, and polarization measurements. Spectroscopic studies on the intrinsic absorption and fluorescence properties of S100 alpha alpha and S100b proteins chemically modified on cysteines-85 alpha and -84 beta with iodoacetamide completed this study. Several arguments suggest that the alkylated S100 proteins undergo conformational changes that are mainly characterized by the destabilization of the quaternary protein structure, which provokes a slow dimer-monomer equilibrium at high protein concentrations and induces total subunit dissociation at low ones. Calcium binding studies on bimane-S100 alpha alpha and -S100b proteins showed that alkylated proteins had a much higher calcium binding affinity than native protein and that the antagonistic effect of KCl on calcium binding was much less pronounced. These results confirmed our previous observations that the affinities of calcium binding sites II alpha and II beta in S100 proteins are highly dependent on protein conformation [Baudier, J., & Gerard, D. (1986) J. Biol. Chem. 261, 8204-8212].

Localization of the high affinity calcium-binding site on tubulin molecule.

J Biol Chem. 1986; 261: 7076-81

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Tubulin is a calcium-binding protein. Two different modes of interaction of calcium with tubulin have been described: a high affinity interaction to one or two binding sites and lower affinity interactions to several other binding sites. In the present study, we have used limited proteolysis of tubulin with trypsin, chymotrypsin, and subtilisin to localize the high affinity calcium-binding sites. Our results indicate that two sites are located in the carboxyl-terminal region of both tubulin subunits, and that tubulin deprived of its carboxyl-terminal region is able to polymerize in the presence of 0.5 mM calcium.

The structures of several calcium-binding proteins are known in considerable detail in both crystalline and solution states. The changes of structure with the binding of calcium, protons, magnesium and hydrophobic molecules are also known. It appears that some calcium-binding structures contain relays of cooperative interaction which run via helices between different parts of these proteins, for example in calmodulin. In others the effect of binding, of for instance calcium, is minimal, as in phospholipase A2. In another group the effect of binding of ions leads to cooperative further binding so that ternary or higher-order complexes are formed, as in the activation of prothrombin. The linking of cellular activities to calcium is thus dependent on several highly developed properties of particular proteins in special environments which have been selected to take advantage of the peculiarities of the calcium ion, namely its particular coordination chemistry and its speed of reaction. The link between calcium, its proteins and activity is not directly to catalysis but is primarily to mechanical properties.

The gene structure of calcium-dependent protease (Ca2+-protease) was determined. It comprises at least 21 exons, and these were assigned to the 4 functional domains of the protease. The protease domain does not show clear correlation between exons and functional units, but the calmodulin-like calcium-binding domain shows strong correlation. Each of the 4 consecutive calcium-binding regions in the C-terminal part of Ca2+-protease is encoded by one exon. This gene structure supports the idea that the 4 calcium-binding regions of calcium-binding proteins such as calmodulin arose by 2 steps of gene duplication.

The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins.

J Biol Chem. 1986; 261: 8761-77

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The structure of bovine intestinal calcium-binding protein (ICaBP) has been determined crystallographically at a resolution of 2.3 A and refined by a least squares technique to an R factor of 17.8%. The refined structure includes all 600 non-hydrogen protein atoms, two bound calcium ions, and solvent consisting of one sulfate ion and 36 water molecules. The molecule consists of two helix-loop-helix calcium-binding domains known as EF hands, connected by a linker containing a single turn of helix. Helix-helix interactions are primarily hydrophobic, but also include a few strategic hydrogen bonds. Most of the hydrogen bonds, however, are found in the calcium-binding loops, where they occur both within a single loop and between the two. Examination of the hydrogen bonding patterns in the calcium-binding loops of ICaBP and the related protein, parvalbumin, reveals several conserved hydrogen bonds which are evidently important for loop stabilization. The primary and tertiary structural features which promote the formation of an EF hand were originally identified from the structure of parvalbumin. They are modified in light of the ICaBP structure and considered as they apply to other calcium-binding proteins. The C-terminal domain of ICaBP is a normal EF hand, with ion binding properties similar to those of the calmodulin hands, but the N-terminal domain is a variant hand whose calcium ligands are mostly peptide carbonyls. Relative to a normal EF hand, this domain exhibits a similar KD for calcium binding but a greatly reduced affinity for calcium analogs such as cadmium and the lanthanide series. Lanthanides in particular may be inappropriate models for calcium in this system.

Calcium performs a unique role in biology, achieving biological effects through highly specific interactions with and modulation of target proteins. It has been proposed that calcium-modulated proteins possess a characteristic, evolutionarily related, binding fold, known as the EF-hand. The high-resolution X-ray structure of alpha-lactalbumin reveals a Ca2+ binding fold that resembles an EF-hand only superficially and presumably has no evolutionary relationship with it. However, there is clear homology with the corresponding loop in c-type lysozyme (the 'parent' molecule of alpha-lactalbumin). This study, at 1.7 A resolution, represents one of the most accurate analyses of a calcium binding protein yet reported.

The enthalpy of calcium binding to frog parvalbumin (Rana temporaria isoenzyme IVb, pI 4.75) has been measured by microcalorimetry. The reaction is exothermic; the heat of the reaction in 100 mM KCl, 50mM Tris, pH 8.0 at 12 degrees C is -19 kJ (mol site)-1 and -33 kJ (mol site)-1 in the presence of 1 mM magnesium. The shape of the titration curve indicates that the properties of the two calcium binding sites are different. The thermodynamic parameters measured for frog parvalbumin are compared with those of related parvalbumins from carp and whiting.

Computer search of calcium binding sites in a gene data bank: use of learning techniques to build an expert system.

Biochimie. 1985; 67: 555-60

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Using a learning set of 28 sequences able to bind calcium (each sequence is 12 residues long), we have built two filters by learning on this set. The first filter uses a pattern-matching technique and the second one takes into account the environment of amino-acids. These two filters have been used to find new calcium-binding proteins in a data bank. The results are discussed.

A partial amino acid sequence for bovine adipose tissue S100 was elucidated by characterization of peptides generated by cyanogen bromide cleavage. The cyanogen bromide peptides were aligned by homology with the bovine brain S100 beta sequence. The results demonstrate that adipose S100 beta is probably identical to brain S100 beta, and suggest that S100 beta is a conserved protein among tissues of the same species.

Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins.

Biochemistry. 1985; 24: 5298-302

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The refinement of the crystal structure of turkey skeletal muscle troponin C at 2.2-A resolution reveals that the two calcium binding loops that are occupied by Ca2+ ions adopt conformations very similar to those of the two homologous loops of parvalbumin and to that of loop III-IV of the intestinal calcium binding protein. This specific fold assures suitable spatial positioning of the Ca2+ ligands. It consists of two reverse turns, one located at each end of the loop, and four Asx turns (a cyclic hydrogen-bonded structure involving an oxygen of the side chain of residue n and the main-chain amide nitrogen of residue n + 2) whenever such a side chain coordinates to the metal ion. The fifth Ca2+ coordination position in both loops of troponin C is occupied by a water molecule that is within hydrogen-bonding distance of an aspartic acid, thus mediating indirect interaction between the cation and the negatively charged carboxylate. The same loop framework is conserved in the two Ca2+ binding loops of parvalbumin and loop III-IV of the intestinal Ca2+ binding protein in spite of the variability in the nature of the side chains at equivalent positions. The disposition of the Ca2+ and of its coordinating water molecule relative to the protein main chain is conserved in all these cases.

Only one major low-Mr calcium-binding protein could be isolated by h.p.l.c. procedures from aqueous extracts of homogenized adult rat skin. This was shown by tryptic peptide mapping and independent amino acid sequence analysis to be identical in all 109 residues with the parvalbumin from rat skeletal muscle. This calcium-binding protein was not in skin epidermis, but was confined to the dermal layer. Skin calcium-binding protein is therefore parvalbumin.

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.

Isolation of mammalian calelectrins: a new class of ubiquitous Ca2+-regulated proteins.

Biochemistry. 1984; 23: 1103-9

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In a new approach to isolating proteins which participate in the Ca2+-dependent regulation of membrane traffic in animal cells, two new Ca2+-binding proteins (Mr 67 000 and 32 500) have been identified in and purified from bovine liver, brain, and adrenal medulla. These proteins specifically and reversibly bind to chromaffin granule membranes at low Ca2+ concentrations (half-maximal binding at 5.5 microM Ca2+) and greatly potentiate the Ca2+-induced aggregation of these membranes at higher concentrations (above 10 microM). In the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, the isolated proteins have Stokes radii of 3.40 nm (Mr 67 000) and 2.53 nm (Mr 32 500) as estimated by gel filtration and therefore occur as monomers. They are slightly acidic proteins with pI's of 5.85 and 5.60. In bovine tissues, both proteins and a third protein of Mr 35 000 cross-react immunologically with each other and with Torpedo calelectrin (Mr 34 000) and are therefore identified as mammalian calelectrins. In all tissues of Torpedo marmorata tested, only a single molecular mass form of calelectrin exists, whereas multiple forms of calelectrin exist in mammalian tissues, indicating gene duplication during evolution. We suggest that the evolutionary conservation and diversification, the high tissue concentrations, and the Ca2+-specific interactions of the calelectrins make them candidates for Ca2+-dependent regulators of membrane events in animal cells.

The amino acid sequence of the beta chain of shrimp SCP has been determined. It is composed of 192 amino acid residues and is acetylated at the N-terminus. The molecular weight was determined to be 21,960. The sequence difference with the aB chain of the same shrimp, of which the sequence was determined previously (J Biochem. (1984) 95, 1603-1615), is 19% (37 non-identical residues out of 192 residues). The shrimp SCPs have three EF-hand type Ca2+ binding sites, however, from comparison with the amino acid sequences of SCPs of scallop (Takagi et al., Biochim. Biophys. Acta in press) and of sandworm (Kobayashi et al., manuscript in preparation), it is reasonable to think that SCP originally had four Ca2+ binding sites, and in the case of shrimp SCPs, one of them (site IV) may have lost the affinity to Ca2+ on amino acid replacements during evolution.

Measurement of facilitated calcium diffusion by a soluble calcium-binding protein.

Biochim Biophys Acta. 1984; 773: 91-8

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The flux of calcium through an aqueous compartment was determined in a flow-dialysis cell in which two dialysis membranes separated the middle aqueous compartment from two outer compartments. The contribution of convection to the total calcium flux was large but could be removed by addition of 1% agar. The flux of calcium through the gelled aqueous compartment agreed with theoretical expectations. The self-diffusion coefficient for calcium from these results was calculated to be 0.81 X 10(-5) cm2 X s-1. Carp parvalbumin significantly enhanced the calcium flux at 2.3 X 10(-6)M free calcium. The calcium flux increased linearly with parvalbumin concentration. These observations are consistent with the hypothesis that the overall unidirectional calcium flux J is the sum of free calcium diffusion and protein-calcium diffusion: J = D[Ca] + D'[CaPr]. The value of D', the self-diffusion coefficient for parvalbumin, was calculated from the flux data to be 13.7 X 10(-7) cm2 X s-1.

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

Biochim Biophys Acta. 1984; 791: 164-72

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We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Trigger activity implies the transfer of the energy of a signal to some amplified (energy) response. Actions in cells, from calcium concentration changes to major protein reorganization are discussed here. The changes must be fast, so mobile polymers must be involved. The first step is the calcium on/off binding to its receptor, calmodulin, troponin C or a comparable protein. Calcium binding is to a loop, EF-hand, between helices. The structures and internal mobilities of these proteins are described using nuclear magnetic resonance and the temperature dependence of NMR shifts. It is suggested that these proteins illustrate a general working hypothesis that proteins made from interacting helices as opposed to beta-sheet proteins will have relatively easy internal main chain motions. Loops connecting the helices then provide particularly obvious read-out points, for example of the initial message of calcium binding. These and other regions of loose structure appear to be associated with highly charged sequences. The further transfer of the trigger message is to highly mobile sequences in troponin I, troponin T and tropomyosin.

The sarcoplasmic calcium-binding proteins (SCP) from crayfish, lobster and shrimp myogen have been purified to homogeneity. These proteins exist as dimers and dissociate in the presence of sodium dodecyl sulfate or urea in subunits of 22000 molecular weight. During the last step of purification (DEAE-cellulose chromatography), SCP emerges in three peaks in the ratio of 14:1.5:1 for crayfish, of 7:2:1 for lobster and of 3:2:1 for shrimp. Gel electrophoresis and isoelectrofocusing experiments, under native and denaturing conditions, indicate that among the three SCP isotypes there are only two different polypeptide chains, alpha and beta, which appear in the form of three dimers: alpha 2, alpha beta and beta 2. The alpha and beta subunits differ slightly in polypeptide chain composition as found by amino acid analyses of the crayfish and lobster SCPs, and also by comparison of tryptic peptides for crayfish SCPs. The polymorphism observed in crustacean SCPs, which is increased by their ability to form dimers, contrasts with the situation prevailing among other invertebrate SCPs and vertebrate parvalbumins where only monomeric isotypes are found. Equilibrium binding studies show that all three SCP isotypes from both crayfish and lobster display the same metal-binding properties. They have in their dimeric form six Ca2+-binding sites: two calcium-specific sites, two Ca/Mg sites that interact with positive cooperativity and two Ca/Mg sites that interact with negative cooperativity. Interactions between the two subunits of SCP seem to result in cooperative binding of Ca2+, which in turn may control more efficiently Ca2+ fluxes in crustacean muscle.

1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80 s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.

The effect of magnesium, calcium, and strontium ions on the bovine bone Gla protein were studied by CD spectroscopy, UV difference spectroscopy and the binding of magnesium and calcium ions was studied by equilibrium dialysis. CD measurements indicate an increase of alpha-helix upon addition of alkaline earth ions. UV difference spectra show a change in the environment of a tyrosine residue. Evaluation of the results of all three studies indicate positive cooperativity in binding of calcium and strontium ions and negative or no cooperativity in binding of magnesium ions.

The single phosphoprotein of fetal calf dentin, having a molecular weight of approximately 94,000 and a phosphorus content of 8% (w/w), was examined by 31P NMR spectroscopy. The single resonance at 3.7 ppm at pH 10 and its chemical shift during acid titration established the phosphomonoester nature of the organic phosphorus moiety. During titration of the phosphoprotein with CaCl2 in the presence of inorganic orthophosphate ions, line broadening for the orthophosphate resonance was both phosphoprotein- and calcium-dependent, indicating ternary complex formation. The data indicate that the phosphoprotein of fetal calf dentin binds both calcium and inorganic orthophosphate ions and therefore has the requisite physical chemical properties necessary for it to facilitate the heterogeneous nucleation of a Ca-PO4 solid phase from solution during tissue mineralization.

The present work has demonstrated the utility of the diamagnetic lanthanides lutetium and lanthanum as metal binding probes for a synthetic 13-residue fragment representing calcium binding site 3 of rabbit skeletal troponin C (residues 103-115). The peptide conformation induced by these metals was monitored by the proton magnetic resonance at 270 MHz. The peptide affinity for these rare earths is 50-400 times higher than that for calcium (KLu3+, 1.3 X 10(4) M-1; KLa3+, 1.1 X 10(5) M-1; KCa2+, 3 X 10(2) M-1) which is related to the change in cation charge from 2+ to 3+. The peptide conformation induced by the presence of La3+ generates a different 1H NMR spectrum than the one observed for the lutetium-saturated peptide. Thus, it appears that these metals do not fold the peptide into exactly the same conformation. The resonance shifts observed during the Lu3+ titration are much smaller than those seen in the case of La3+ addition. The fact that lutetium binds less tightly than lanthanum to the peptide may be linked directly or indirectly to the difference in ionic radius between these metals (Lu3+, 0.86 A; La3+, 1.03 A). This may in turn indicate that the peptide primary sequence encodes for some aspects of metal ion specificity. The 1H NMR results also demonstrate that glycine-108 adopts a restricted geometry in the absence of metal such that its two alpha-carbon protons are in different environments which are further affected by the addition of either metal. These observations support the concept that geometric constraints arising from the particular peptide folding pattern near this residue correlate with the highly conserved nature of this site of the EF hand. This position remains occupied by glycine in most EF hand domains with the exception of known distorted calcium binding sites present in intestine calcium binding proteins and S-100.

Using synchrotron radiation at the Frascati storage ring ADONE, the X-ray Absorption Near Edge Structure (XANES) has been applied to determine homologies and modifications of the local structure of the calcium binding sites of troponin C. In all four calcium binding sites, Ca2+ appears to be co-ordinated to carboxyl and carbonyl groups in a characteristic configuration. No structural difference has been found between high and low-affinity sites. A distortion of the Ca2+ site geometry by binding of Mg2+ has been observed. The XANES of parvalbumin has been measured and found to be different from troponin C. A tentative identification of the characteristic XANES spectra of the two different Ca2+ sites in this protein is reported.

Ion binding to calmodulin. A comparison with other intracellular calcium-binding proteins.

Mol Cell Biochem. 1983; 51: 33-54

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Over the past few years calcium has emerged as an important bioregulator. Upon external stimulation, the cell generates a transient Ca2+ increase, which is transformed into a cellular event through a molecular cascade. The first step in this cascade is the binding of calcium to proteins present in the cytosol. These proteins capable of binding Ca2+ under physiological conditions all belong to the same evolutionary family that evolved from a common ancestor. However, they strongly differ in the properties of their calcium binding sites. Calmodulin, the ubiquitous calcium binding protein present in all eukaryotic cells, is very close to the ancestor protein, presents four calcium binding sites which bind calcium, magnesium and monovalent ions competitively and is involved in the triggering of cellular processes. Parvalbumin, another member of the family, is more specialized and found mostly in fast-twitch skeletal muscle. It binds calcium and magnesium with high affinity and seems to be involved in muscle relaxation. On the other hand, troponin C which confers Ca2+ sensitivity to acto-myosin interaction exhibits both triggering and relaxing sites. The study of intracellular Ca2+ binding proteins has shown that calcium binding proteins have evolved from a simple common structure to fulfill different functions.

Demonstration of three distinct calcium-binding sites in villin, a modulator of actin assembly.

J Biol Chem. 1983; 258: 365-9

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Villin, a Ca2+-modulated F-actin-binding protein (95,000 daltons) present in microvillus core filament bundles, has been shown to contain multiple Ca2+-binding sites. 45Ca Hummel-Dreyer chromatography reveals the presence of two rapidly exchanging Ca2+-binding sites with an apparent dissociation constant, Kd, equal to 4.6 X 10(-6) M. Use of the two proteolytically separable domains of the molecule revealed that one site is located on the 90,000-dalton core (apparent Kd = 3.5 X 10(-6) M) while the second site is provided by the 8,800-dalton headpiece fragment (apparent Kd = 7.4 X 10(-6) M). In addition villin displays a further very slowly exchanging or nonexchangeable high affinity Ca2+-binding site, which is situated in the core domain. Secondary structural predictions and a comparison of the amino acid sequence of headpiece with other known Ca2+-binding proteins indicates one region suggestive of a Ca2+-binding site, although headpiece seems not to exhibit a classical "EF-hand" Ca2+-binding structure.

The changes in heat capacity and entropy of troponin C induced by calcium binding.

J Biochem (Tokyo). 1982; 92: 1505-17

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We have used the method of enthalpy titration to analyze the structural changes of troponin C caused by calcium binding. Successive additions of calcium to metal-free troponin C in a microcalorimeter cell result in at least three distinguishable transitions. Analysis of the results has shown that troponin C has at least three classes of calcium binding sites; one site of highest affinity (binding constant, 10(8)-10(10) M-1), one site of next highest affinity (binding constant, 10(6)-10(7) M-1) and two low affinity sites (binding constant, 10(5)-10(6) M-1). Titrations of troponin C with calcium at various temperatures have shown that calcium binding causes large changes in the heat capacity of troponin C. Following the method of Sturtevant (1977), the magnitudes of the hydrophobic and intramolecular vibrational contributions to the heat capacity and entropy changes of troponin C on calcium binding have been estimated. In Mg-free solutions, calcium binding to the 1st site of highest affinity gives rise to a strong hydrophobic effect and to a tightening of the molecular structure. In contrast, calcium binding to the 2nd site of next highest affinity gives rise to a strong hydrophobic effect in the reverse direction and to a "softening" of the structure. Calcium binding to the two low affinity sites has a moderately strong hydrophobic effect and also causes a moderate tightening of the structure. These results are in many respects similar to those obtained with proton magnetic resonance spectroscopy by Levine et al. (1977). These studies are mutually complementary. When 1 mM magnesium is present the changes caused by calcium binding to the two high affinity sites are greatly altered, whereas those involving the two low affinity sites are not much affected. The moderate tightening of the structure which is caused by calcium binding to the two low affinity sites, and which is seen both in the absence and presence of magnesium, is most likely to be involved in the regulation of contraction.

[Characterization of the three calcium binding proteins in the human placenta (author's transl)]

Nippon Naibunpi Gakkai Zasshi. 1982; 58: 662-78

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Calcium binding protein (CaBP) has been reported to be involved in absorbing calcium in the duodenum. There is some evidence to show that active transport of calcium ions occurs from mother to fetus in the human placenta. The presence of CaBP in the human placenta was examined according to Taylor and Wasserman's method, extracting the vitamin D-dependent CaBP in the duodenum, and the character of CaBP was then studied. 38,000 X g supernatant of the villous homogenate of the human placenta was heated at 60 degrees C for 5 mins to neutralize the calcium binding activity resulting from the blood. The fractions containing CaBP were separated and partially purified on Sephadex G-100 and DEAE cellulose columns. The CaBP fractions were further purified by preparative agar electrophoresis. The Chelex-100-resin method was used for the detection of calcium binding activity in the eluates and for calculation of Kd values and of calcium binding sites. The molecular weights were determined by electrophoresis of SDS-polyacrylamide gel. Three CaBPs were found in the human placenta. The molecular weight, Kd (micro M) and the binding activity (Ca mol/CaBP mol) of the three placental CaBPs were CaBP-Peak I: 82,000, 0.64, 0.52; Peak II: 12,000, 0.67, 1.10; Peak III: 8,500, 0.75, 2.08, respectively. The tyrosine residue of CaBP has been shown to act as an important binding site in that removal of calcium ions from CaBP generates a blue-shifted phenomenon in the ultraviolet difference spectrum between 270 and 300 nm. However no difference spectrum was observed with the placental CaBPs. This result was further confirmed by amino acid analysis which showed that none of the placental CaBPs contained tyrosine, tryptophan, half-cystine and proline, which are usually found in most proteins. The circular dichronism (CD) spectrum of CaBP-Peak I in the far ultraviolet range showed two negative bands at 222 and 207 nm (alpha-helix structure), and removal of calcium ions caused no difference spectrum. CD spectra of CaBP Peak II and III in the far ultraviolet range revealed random coil structures in the presence and absence of bound calcium ions. These findings indicate that the calcium binding mechanism of the placental CaBPs should be different from that of the others. In this study, three kinds of human placental CaBP newly isolated were characterized according to molecular weights, Kd values and binding activities and clarified as having an amino acid composition quite different from the CaBPs already reported.

The role of calcium as an intracellular second messenger is now widely recognized. Upon stimulation of a eukaryotic cell, its cytosolic concentration increases 100 fold from pCa 7 to pCa 5. Calcium effects within the cell are mostly mediated through binding of the ion to low molecular weight proteins that form an evolutionary family including parvalbumin, calmodulin, troponin C, the alkali and regulatory light chains of myosin, the S-100 protein and the vitamin D-dependent intestinal calcium-binding protein. The structural, evolutionary and functional unit of each of these proteins is the calcium-binding domain, made of a 12-residue binding loop flanked on each side by a ca 12-residue long alpha-helix. Calcium binding proteins differ in the number of domains and in the Ca2+-binding properties of the domains. Parvalbumin exhibits two high affinity Ca2+-Mg2+-binding sites involved in the relaxation phase of fast skeletal muscle. In contrast, calmodulin and troponin C exhibit low affinity Ca2+-specific sites that trigger Ca2+-dependent effects. Calcium binding to calmodulin is sequential and ordered and results in a conformational change that enables the protein to interact through a hydrophobic patch with the numerous calmodulin-dependent enzymes. Calmodulin is highly conserved, ubiquitous and multifunctional. It is responsible for the Ca2+ control of cell motility, of energy production through the glycogenolytic pathway, and of the modulation of messengers, amongst which are Ca2+ itself, cAMP and neurotransmitters.

Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the alpha helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the alpha-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) can be mimicked by other cations. Tyr fluorescence, UV difference spectra, and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.

Proton magnetic resonance spectroscopy has been employed to study the solution conformation of three cleavage fragments of troponin-C, each containing a single Ca(II)-binding site and corresponding to different regions in the primary sequence; viz. CB8 (residues 46-77), CB9 (residues 85-134) and TH2 (residues 121-159). Although all three peptides lack a well-defined tertiary fold in the absence of metal ions, several spectral features indicate the presence of local conformational constraints in each apo-peptide. Ca(II) binding led to spectral changes consistent with increased restriction of backbone motility and the adoption of a more compact conformation. Studies using paramagnetic ions as conformational probes support current views concerning the nature of the ligands at the metal binding sites. The nature and kinetics of the structural influence of metal binding suggest that the conformational constraints existing in the CB8 apo-peptide provide an adequate Ca(II)-binding configuration. In contrast, the CB9 and TH2 peptides exhibit spectral changes consistent with an increased local structure in the region of helix E (residues 94-102) in the case of CB9 and helix H (residues 148-159) in the case of TH2. In CB9, conformation changes also appear to be transmitted to a portion of the sequence (residues 87-93) preceding helix E, a putative site of interaction between troponin-C and troponin-I. These data are discussed with reference to the contribution of long-range (interdomain) interactions within troponin-C and the modulation of troponin subunit protein-protein interactions by Ca(II) binding.

The Ca2+-binding properties and amino acid compositions of two calcium-binding proteins (mCaBP-3 and mCaBP-4) purified from bovine milk were studied. mCaBP-3 was identified as a Ca2+-bound type and mCaBP-4 as a Ca2+-free type by means of ion-exchange chromatography on a DEAE-Sephadex A-25 column. In polyacrylamide gel disc electrophoresis, both mCaBP-3 and mCaBP-4 had the same mobility of Rf = 0.73 and the addition of 5 mM CaCl2 to the electrode buffer decreased the mobility from Rf = 0.73 to Rf = 0.49. mCaBP-3 and mCaBP-4 consisted of 120 and 122 amino acid residues, respectively. The molecular weights were 13,758 and 13,967, respectively. The amino acid compositions of the two milk CaBPs very closely resembled each other. Both milk CaBPs were rich in aspartic acid, glutamic acid, leucine and lysine, but did not contain trimethylated lysine and amino sugar. An interesting feature is that each milk CaBP contained eight cysteine sulfone and three tryptophan residues per molecule. From these results, it is suggested that mCaBP-3 and mCaBP-4 are identical protein and that mCaBP-3 is formed from mCaBP-4 by means of a conformational change by binding of Ca2+. Thus, mCaBP-3 is a holoprotein and mCaBP-4 is an apoprotein. Furthermore, it is suggested that milk CaBP is different from calmodulin, troponin C and vitamin D-dependent calcium-binding protein.

The conformation of troponin C (TN-C) isolated from the white muscle of pike (Esox lucius), in the Ca2+ and metal-free states, was studied by circular dichroism, absorption difference spectroscopy, solvent perturbation difference spectroscopy, intrinsic fluorescence, thiol titration, and 1H nuclear magnetic resonance spectroscopy. In addition, the molecular weight of the protein was determined by sedimentation equilibrium and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The composition of the protein was established by amino acid analysis. The resulting data were compared with those from the widely studied analogue isolated from rabbit skeletal muscle. The results indicate near equivalence in many of the properties of pike and rabbit TN-C, such as molecular weight, the magnitude of the calcium-induced conformational change, and urea- or thermal-induced denaturability. However, the pike protein has five additional potential carboxyl groups, and there is good evidence from NMR, solvent perturbation, and fluorescence studies for the presence of a buried tyrosine residue in the apo state.

The soluble calcium-binding protein from muscle of the sandworm, Nereis virens.

J Muscle Res Cell Motil. 1981; 2: 225-38

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Fast-acting muscle of the sandworm, Nereis virens, contains one soluble calcium-binding protein having a molecular weight close to 17 000 and occurring in the muscle at a concentration of approximately 0.1 mM. The protein binds two Ca2+ at equivalent sites with dissociation constant Kd = 6.4 X 10(-7) M. Its N-terminal amino acid is blocked by an N-acetyl group whereas glycine is the C-terminal residue. The comparison of the tryptic peptide map of this protein with those of the soluble calcium-binding protein from crayfish muscle, bovine brain calmodulin and rabbit skeletal muscle troponin C suggests that all of these proteins are homologous. Sandworm calcium-binding protein therefore belongs to the so-called cytosolic calcium EF-hand family. This protein is presumably the functional counterpart of vertebrate parvalbumin acting as soluble relaxing factor.

Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium.

Cell. 1981; 26: 447-54

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Compaction, a process of cell-cell adhesion between mouse blastomeres or between embryonal carcinoma (EC) cells requires calcium ions. A decompaction effect similar to that observed in the absence of Ca2+ is triggered by Fab fragments of rabbit anti-EC IgG. This effect occurs through the recognition of a specific cell-surface glycoprotein named uvomorulin. An 84,000 dalton fragment of uvomorulin (UMt) has been previously extracted by trypsin from EC cell membranes and purified. WE present evidence that effects of Ca2+ on compaction are transmitted through conformational changes in uvomorulin. First, Ca2+ protects UMt from further proteolysis by trypsin. Mn2+ and Sr2+ have similar effects, whereas this protection is reversed by La3+. Second, UMt can bind the monoclonal antibody De1 only in the presence of Ca2+ (half-binding at 10(-5) M Ca2+). This antigenic exposure also takes place in the presence of Mn2+ or Sr2+ and is reversed by La3+. Third, metal ions (Ca2+, Mn2+, Sr2+) that promote trypsin resistance and recognition by DE1 are found to trigger the compaction of morulae and EC cells. Metal ions (La3+) that reduce trypsin resistance and affinity for DE1 result in decompaction.

Allergenic synthetic peptide corresponding to the second calcium-binding loop of cod allergen M.

Scand J Immunol. 1981; 14: 207-11

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A peptide with the sequence of the second calcium-binding loop (EF loop) of cod Allergen M was synthesized by automatic solid-phase technique. The synthetic peptide corresponded to residues 88-103 of the known primary structure of Allergen M. The immunochemical reactivity of this loop, previously demonstrated for the overlapping enzymic fragments, was confirmed by using the synthetic preparation. The purified hexadecapeptide was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals, in both in vivo and in vitro tests systems. It could also bind rabbit anti-Allergen M, as shown by rocket line immunoelectrophoresis and quantitative precipitation inhibition techniques. The findings emphasized that the immunological reactivity of the synthetic peptide (88-103) was compatible with a monovalent haptenic function: blocking and not eliciting allergic reactions.

The effect of Ca2+ binding on the 270-MHz proton nuclear magnetic resonance spectrum of bovine cardiac troponin C (cTnC) has been examined. Assignment of resonances in the aromatic spectral region to tyrosine residues 10, 111, and 150 has been made for apo-cTnC and calcium-bound cTnC on the basis of decoupling experiments, pH titrations, temperature-induced changes, and gadolinium broadening experiments. The sequence homology which these tyrosine residues display with residues in two previously studied proteins, rabbit skeletal troponin C (sTnC) [Seamon, K. B., Hartshorne, D. J., & Bothner-By, A. A. (1977) Biochemistry 16, 4039] and bovine brain calmodulin [Seamon, K. B. (1980) Biochemistry 19, 207], was also used in assignments. High-affinity calcium binding (up to 2 mol/cTnC) causes large alterations in the environments of tyrosines-10 and -150, indicating that the N terminus is probably buried in the protein interior. The evidence suggests that the environment of tyrosine-150 in calcium-saturated cTnC must closely resemble that of tyrosine-138 in calmodulin in that it experiences the hydrophobic core of the protein. However, there is no similarity between these environments in the apoproteins. Dramatic alterations in phenylalanine resonances are seen during the binding of the third mole of calcium, corresponding to filling the sole low affinity site. Comparison of the spectral calmodulin reveals many structural similarities which stem from their high degree of primary sequence homology.

The tryptophan-containing subunit (alpha-subunit) of bovine brain S-100 protein was purified from a S-aminoethyl derivative of S-100a protein, and its amino acid sequence was determined. The alpha-subunit contained 93 residues, including one tryptophan, and had a molecular weight of 10,400. The sequence shows an extensive homology (58% identity) to the sequence of another "tryptophan-free" subunit (beta-subunit) found in both S-100a and S-100b protein, and has a calcium binding site characteristic of the "E-F hand" proteins, such as calmodulin or troponin C. The tryptophan residue is located at position 90 which is presumably adjacent to the C-terminal end of the alpha-helix following the calcium binding loop, and thus appears likely to serve as a specific probe in structure-function studies of S-100 protein.

A protein in the bone matrix that retains calcium is described. This protein seems to start the ossification process. The reaction occurs in several steps from high energy phosphorylation to the formation of aggregates and calcium phosphate.

Calcium-ion-binding activity in human small-intestinal mucosal cytosol. Purification of two proteins and interrelationship of calcium-binding fractions.

Biochem J. 1981; 197: 55-65

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Ca2+-binding activity was investigated in human small-intestinal mucosal cytosol. Binding was detected in fractions with molecular weights of 28000 and about 900000, as determined by gel filtration. No binding was found at molecular weight 12000-13000 (the molecular weight of calcium-binding protein in lower mammalian species) until the cytosol had been subjected to a hollow-fibre-filtration step. The appearance of Ca2+-binding at molecular weight 12000-13000 was associated with a decline in the 28000-mol.wt. calcium-binding fraction. The 12000-13000-mol.wt. fraction contained two distinct calcium-binding proteins. One of these proteins had properties similar to those of pig calcium-binding protein. Antiserum to this protein reacted against the 28000-mol.wt calcium-binding fraction in cytosol from human small-intestinal mucosa and from human kidney. An immunoassay method for one of the calcium-binding proteins was established. In normal duodenal mucosa the concentration was 915 micrograms/g and in the ileum it was 443 micrograms/g of mucosa. A subject with hypercalcaemic sarcoidosis had 1200 micrograms/g of mucosa in the jejunum, and a subject with an undetectable concentration of plasma 25-hydroxycholecalciferol had concentrations of calcium-binding protein in the mucosa similar to those found in normal subjects.

Laser photo-CIDNP 1H NMR experiments were performed with rabbit skeletal troponin-C (sTn-C), bovine cardiac troponin-C (cTn-C), and bovine brain calmodulin to study the exposure of histidine and tyrosine residues. In cTn-C, tyrosine residues, 5, 111, and 150 were exposed in the apoprotein, becoming buried as Ca2+ was bound. A similar phenomenon was observed for tyrosine residues 10 and 109 of sTn-C. In calmodulin, only tyrosine-99 was accessible in the apoprotein. The lack of exposure of tyrosine-138 observed with this technique correlates with the buried nature of this residue implied by other criteria. In 6 M urea each of the apoproteins were observed to be unfolded from the standpoint of the tyrosine environments. A large tyrosyl CIDNP effect was obtained for each protein which decreased as Ca2+ was bound, with a stoichiometry of one metal ion per protein. This was correlated for cTn-C with the appearance of "native" resonances representing tyrosine residues 111 and 150 in Ca2+-saturated cTn-C, also with a stoichiometry of one. Analysis of our NMR findings, in the light of other spectroscopic and model building studies on these systems, suggests that the sole high-affinity Ca2+ binding site of cTn-C and sTn-C remaining in 6 M urea is site IV.

Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes.

Biochemistry. 1981; 20: 3890-7

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The acid stability of calmodulin has been used to devise a rapid and efficient method of decalcification based on trichloroacetic acid precipitation. Study of the competitive binding of K+, Mg2+, and Ca2+ to the Ca2+-binding sites of calmodulin has allowed determination of the intrinsic binding constants of each of the three cations for the four Ca2+-binding sites. The data are compatible with an ordered binding of Ca2+. If the Ca2+ sites are labeled A, B, C, and D starting at the NH2 terminus, the order of binding is postulated to be B, A, C, and D. The ordered binding properties support the suggestion that calmodulin translates quantitative Ca2+ signals into qualitatively different cellular responses.

The primary structure of a salivary calcium-binding proline-rich phosphoprotein (protein C), a possible precursor of a related salivary protein A.

J Biol Chem. 1980; 255: 5943-8

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The complete primary structure of a calcium-binding "proline-rich phosphoprotein" named salivary Protein C was determined from peptides obtained by enzymatic and chemical cleavages of the protein. The protein consists of a single polypeptide chain of 150 residues. It contains the entire primary structure of a previously isolated salivary Protein A in its NH2-terminal 106 residues. The COOH-terminal 44 residues consist mostly of glycine, glutamine, and proline, including a hexaproline sequence, but no polyproline structure could be detected by CD spectroscopy. There is extensive repetition of sequences in the protein, suggesting gene multiplication and recurrent folding. Comparison of the primary structure of salivary Proteins A and C with known protein sequences indicate that the salivary proteins constitute a new family. A mouse submaxillary protease will cleave salivary Protein C between residues 106 and 107 only, giving rise to salivary Protein A and a 44-residue COOH-terminal peptide. This cleavage and the sequence data suggest that salivary Protein C may be a precursor of salivary Protein A.

The Ca2+ indicator, arsenazo III, binds to subcellular fractions of rabbit skeletal muscle with sufficient affinity that in living muscle containing 1--2 mM arsenazo III, the estimated free arsenazo III concentration is only 50--200 microM; 80--90% of the bound arsenazo III is associated with soluble proteins. The binding of arsenazo III to soluble proteins decreases the optical response of the dye to Ca2+; this is due to a decrease in the affinity of the protein-bound dye for Ca2+. Approximately half of the bound arsenazo III is released from the particulate fraction and soluble proteins upon addition of 5 mM Ca2+, suggesting that the Ca-arsenazo complex has lower affinity for the protein binding sites than the free dye. The Ca2+ binding to the soluble protein fraction of rabbit skeletal muscle is attributable largely to its parvalbumin content.

Effects of prednisolone on the biochemical indices of blood serum, ash content in bones, calcium absorption and calcium-binding protein (CaBP) in the intestinal mucosa were studied. The oral administration of prednisolone (10 mg/kg) for 2 weeks reduced CaBP content and calcium absorption to the level typical for D-avitaminosis. The oral administration of 1 alpha-hydroxycholecalciferol (390 pmoles/day) in combination with prednisolone for a week returned calcium absorption and CaBP to the control value.

The sequence domains that contribute to the surfaces of contact between Troponin-C and the other regulatory protein subunits of skeletal muscle troponin are proposed on the basis of data obtained by proton magnetic resonance and other physicochemical studies on the interaction with Troponin-I of both Troponin-C and its peptide fragments. Marked sequence homology in Troponin-C from various species is found for the residues involved in subunit linkage. The role of the recognition sites is discussed.

Calmodulins have been purified from porcine, rabbit, rat, and chicken brains and their structural and functional properties compared to those of the bovine brain protein whose complete amino acid sequence has been elucidated. No major differences were detected in the amino acid compositions and tryptic peptide maps of these five proteins. All calmodulins lacked tryptophan and cysteine and contained 1 mol of N epsilon-trimethyllysine and histidine per mol of protein. Bovine, porcine, rabbit, rat, and chicken brain calmodulins comigrated on polyacrylamide gels run under a variety of conditions in the presence and absence of denaturants. All brain calmodulins gave identical profiles for the calcium-dependent activation of "activatable" bovine brain 3',5'-cyclic nucleotide phosphodiesterase. In addition, they formed calcium-dependent complexes with rabbit skeletal muscle troponin I and the electrophoretic mobilities of the complexes were identical with one another and similar to the corresponding complex between troponin I and troponin C. These studies more fully define what is a calmodulin, demonstrate that calmodulin is a relatively invariant constituent of vertebrate brain, and indicate that calmodulin structure and function have been highly conserved throughout vertebrate evolution.

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

Rat mastocyte plasma membrane possess two major classes of calcium binding sites which have widely different affinity. At pH 7.5 the low affinity sites can bind about 88 nmol of Ca2+ per milligram of membrane protein and are half-saturated at 125 micro M Ca2+, whereas the high-affinity sites bind about 12 nmol of Ca2+ per milligram of membrane protein and are half-saturated at 5.5 micro M Ca2+. Membrane phospholipids and neuraminic acid residues account for approximately 87% of calcium binding.

We present the data required to establish the complete amino acid sequence of bovine brain modulator protein, the multifunctional calcium-dependent regulatory protein. Bovine brain modulator protein contains 148 amino acid residues and has a molecular mass of 16,680 daltons. The protein commences with an acetylated alanyl residue in accord with the previous report that its NH2 terminus was blocked. The single residues of histidine and trimethyllysine occur at positions 107 and 115, respectively, in a region of the linear sequence implicated by other studies as important for calcium-dependent modulator protein-enzyme interactions. The sequence of bovine brain modulator protein demonstrated here is closely related to those of muscle troponin Cs, as originally suggested from considerations of the similarities in calcium binding and functional and physicochemical properties of these proteins (Watterson, D.M., Harrelson, W.G., Jr., Keller, P.M., Sharief, F., and Vanaman, T.C. (1976) J. Biol. chem. 251, 4501-4513). The linear amino acid sequence of bovine brain modulator protein is composed of four internally homologous sequences or domains, each of which contains the appropriate amino acids arranged so as to form a helix-loop-helix, calcium-binding structure. The high level of internal homology of bovine brain modulator protein and its relationship to the other members of the calcium-binding protein superfamily provide convincing evidence that 1) it arose early in the evolution of these related proteins and 2) it was formed by two successive tandem duplications of a gene encoding a small, single domain ancestral precursor. Comparison with the nearly complete sequences of the bovine uterus and rat testis modulator proteins reported by other laboratories indicates that this ubiquitous calcium-dependent regulatory protein does not occur in tissue-specific forms, commensurate with the proposed function of modulator protein as a mediator of calcium-second messenger function in eukaryotic cells.

Static fluorescence titration and fluorescence stopped-flow kinetic experiments o N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide-labeled troponin C showed that the molecular kinetic mechanism of the local conformational change induced by the rapid Ca2+ binding and removal reactions with the high affinity Ca2+-binding sites (sites III and IV) can be explained by the following scheme: [Formula: see text]. Numerical constants in this scheme were determined in this work.

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a kobs of 0.012 sec-1. Kinetic measurements were used to calculate the association constant, Ka, for calcium binding to concanavalin A of 2.7 x 10(4) M-1, in reasonable agreement with values obtained by equilibrium methods.

Calcium-binding proteins in serum of chickens: vitellogenin and albumin.

Poult Sci. 1980; 59: 874-9

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The Ca-binding proteins of hen serum were resolved by gel filtration in 45Ca buffer and were named CaBP(1) and CaBP(2). The major calcium-binding protein, CaBP(1), had a molecular size of 6.0 X 10(5) daltons and appears to be vitellogenin. Deeley et al. (1975) described vitellogenin as the precursor of lipovitellin and phosvitin, the Ca-binding proteins of egg yolk. The present paper demonstrates that "native" vitellogenin is a calcium-binding protein in the serum of laying chickens. The CaBP(2) co-eluted with and was immunologically identical to chicken serum albumin which also bound 45Ca. Data from elution profiles and phosphorus assays indicated that CaBP(1) and CaBP(2) were different from phosvitin and the Ca-binding proteins of the duodenum and uterus.

Fusion of phospholipid vesicles with a planar phospholipid bilayer membrane that contains a calcium-binding protein appears to mimic the essential aspects of cytoplasmic-vesicle fusion with plasma membranes (exocytosis) in that (i) there is a low basal rate of fusion in the absence of Ca2+, (ii) this basal rate is enormously increased by micromolar (approximately 10 microM) amounts of Ca2+, and (iii) this rate is not increased by millimolar Mg2+. Essential to this process is an osmotic gradient across the planar membrane, with the side containing the vesicles hyperosmotic to the opposite side. Similar osmotic gradients or their equivalent may be crucial for biological fusion events.

Calcium binding by two proteins, vitellogenin and albumin, was measured in serum of a line of hens producing thick (THK) and a line of hens producing thin shells (THN) as well as a line of commercial hens using gel filtration in 45calcium buffer. Vitellogenin was quantitated in serum of THK and THN using a radial immunodiffusion test. Levels of serum calcium were measured and related to the above mentioned parameters. The binding of vitellogenin was significantly greater in 13 THK than 13 THN (672 vs. 508 cpm/ml X 10(-3), but binding by albumin was not significantly different between the two lines (386 vs. 344 cpm/ml X 10(-3). Binding in 11 commercial hens was similar to that for THN. The THK had significantly greater levels of diffusible, non-diffusible, and total serum calcium than THN. Significant positive correlations between total serum calcium and vitellogenin binding were found in THK (.77) and THN (.81) as well as in the commercial hens (.64). Like vitellogenin binding, levels of vitellogenin were significantly greater in THK than THN (4.0 vs. 1.8 mg/ml). These results suggest that in addition to having more diffusible and non-diffusible serum calcium, THK have more calcium binding and more vitellogenin to perform the binding function than THN.

Sodium-23 nuclear magnetic resonance as an indicator of sodium binding to troponin C and tryptic fragments, in relation to calcium content and attendant conformational changes.

Eur J Biochem. 1980; 105: 289-95

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The relaxation rate enhancements of the 23Na nuclei for NaHCO3 solutions of troponin C and its tryptic peptides TR-1 and TR-2 indicate true binding of Na+ ions to these biomolecules. The low-affinity sites I and II of TR-1 and troponin C are the sites of competitive Na+/Ca2+ binding, below one calcium ion per molecule, with log KNa approximately 2. At low calcium content Na+ ions bind to TR-2 and to troponin C non-competitively with Ca2+ ions; binding of Ca2+ ions to the high-affinity sites III and IV allosterically affects the binding of the Na+ ions: even when sites I and II, located on TR-1 or sites I, II, III, IV of troponin C, are saturated with Ca2+ ions, Na+ ions continue to bind weakly at secondary binding sites.

Calcium-binding ribonucleoprotein in developing chick embryo brain: its relationship to the ontogenesis of electrical activity.

Int J Neurosci. 1980; 11: 195-200

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A calcium-binding ribonucleoprotein, specific to brain, has been shown to increase in quantity with the increase in the age of chick embryo, attaining a plateau around day 12. The increase has a correlation with the onset and maturation of the electrical activity in the developing chick embryo brain.

The purification of a unique calcium-binding protein from Morris hepatoma 5123 tc.

Biochim Biophys Acta. 1980; 621: 296-304

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A heat-stable Ca2+-binding protein was purified to homogeneity from Morris hepatoma 5123 tc. It had an apparent molecular weight of 11 000, and isoelectric point of 3.9, and bound two atoms of calcium per molecule of protein. The spectral and amino acid analysis indicated the tumour protein to be similar to the parvalbumins. This protein has been shown to be absent in liver.

The effect of temperature on some calcium-binding properties of troponin C and calmodulin.

Can J Biochem. 1980; 58: 683-91

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Some calcium-binding properties of skeletal and cardiac troponin C (TnC) have been measured as functions of temperature employing several physical and spectroscopic techniques. The degree of exposure of the tyrosine residues in brain calmodulin has also been determined by a new approach. Circular dichroism thermal unfolding profiles have been established for the three cases: metal-free protein, high-affinity sites filled, and fully saturated. In addition some thermodynamic parameters have been calculated for these reversible melting process. It was found that the calcium-binding parameters n and K, where n is the fraction of the total conformational change and K is the apparent association constant, for both skeletal and cardiac TnC, did not vary significantly over the temperature range 10-38 degrees C, but at 50 degrees C differences became quite apparent, dramatically so in the case of the skeletal protein. The technique of thermal perturbation difference spectroscopy was applied to determine the degree of exposure of aromatic chromophores for the TnC(s) and calmodulin in the absence and presence of calcium. For skeletal TnC and calmodulin the results were in good agreement with previous observations, but the reduced degree of exposure of the tyrosine residues in cardiac TnC, in the absence of Ca2+, was contrary to the earlier work. Calcium-induced difference absorption spectra have been measured for the TnC(s) over the temperature range 10-70 degrees C. Cardiac TnC showed greater heat stability than its skeletal counterpart, in terms of the rate and the amount of change of the difference spectral maxima.

Purification and structural properties of gelsolin, a Ca2+-activated regulatory protein of macrophages.

J Biol Chem. 1980; 255: 9490-3

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We describe the purification procedure and some of the physiochemical properties of gelsolin, a major Ca2+-dependent regulatory protein of actin gel-sol transformation in rabbit lung macrophages. Gelsolin accounts for the majority of Ca2+ control of actin gelation in macrophage extracts. It is a single polypeptide chain with an average molecular weight of 91,000 a Stokes radius of 44 A, a sedimentation coefficient (s20(0),w) of 4.9 S, an isoelectric point of 6.1, and a frictional ratio of 1.43. Gelsolin binds 2 mol of Ca2+ with high affinity (Ka 1.09 X 10(6) M-1) in the presence of 0.1 M KCl and 2 mM MgCl2.

Evolution of homologous physiological mechanisms based on protein sequence data.

Comp Biochem Physiol B. 1979; 62: 1-5

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1. Genetic duplications can give rise to homologous physiological mechanisms that include structurally related protein components. There are many such examples of related proteins within the human body. 2. Evolutionary histories showing the origins and subsequent divergences of these distantly related proteins can be derived from the protein sequences and correlated with the functional characteristics of these proteins. 3. The hormones related to glucagon provide an example of homology of physiological mechanisms and emergence of new functions subsequent to gene duplications. 4. The proteins related to troponin C illustrate the participation of distantly related proteins in the same mechanism (muscle contraction), the relationship of proteins characteristic of a specialized tissue to proteins found in all eukaryote cells, and the correlation of genetic duplications with the evolutionary appearance of different types of muscle.

1H NMR and ORD were used to characterize the respective variations of tertiary structure and secondary structure of parvalbumins with calcium content ((Pa(O), without calcium and PaCa2 calcium saturated) and temperature. It has been observed that the tertiary structure can be lost without significant variation of the helical content. Cooperative binding of calcium to Pa(O) has been shown by NMR spectroscopy under low ionic strength conditions and at neutral pH. The present study shows that the calcium binding affinity of parvalbumin is dependent on the tertiary structure. Calcium binding and calcium release functions of parvalbumins in the muscle may be controlled by their tertiary structure.